RESUMO
BACKGROUND: For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood. METHODS: GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses. RESULT: Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex. CONCLUSION: PKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG.
Assuntos
Glucose-6-Fosfato Isomerase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Neoplasias/metabolismo , Piruvato Quinase/metabolismo , Animais , Carcinoma de Ehrlich/metabolismo , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica , Glucose-6-Fosfato Isomerase/química , Glucose-6-Fosfato Isomerase/genética , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/genética , Espectrometria de Massas , Camundongos , Neoplasias/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Aldeído Pirúvico/farmacologia , Piruvato Quinase/química , Piruvato Quinase/genéticaRESUMO
The creatine/creatine kinase (CK) system plays a key role in cellular energy buffering and transport. In vertebrates, CK has four isoforms expressed in a tissue-specific manner. In the process of creatine biosynthesis several other important metabolites are formed. The anticancer effect of creatine had been reported in the past, and recent literature has reported low creatine content in several types of malignant cells. Furthermore, creatine can protect cardiac mitochondria from the deleterious effects of some anticancer compounds. Previous work from our laboratory showed progressive decrease of phosphocreatine, creatine and CK upon transformation of skeletal muscle into sarcoma. It was convincingly demonstrated that prominent expression of creatine-synthesizing enzymes L-arginine: glycine amidinotransferase and N-guanidinoacetate methyltransferase occurs in sarcoma, Ehrlich ascites carcinoma and sarcoma 180 cells; whereas, both these enzymes are virtually undetectable in skeletal muscle. Creatine transporter also remained unaltered in malignant cells. The anticancer effect of methylglyoxal had been known for a long time. The present work shows that this anticancer effect of methylglyoxal is significantly augmented in presence of creatine. On creatine supplementation the effect of methylglyoxal plus ascorbic acid was further augmented and there was no visible sign of tumor. Moreover, creatine and CK, which were very low in sarcoma tissue, were significantly elevated with the concomitant regression of tumor.
Assuntos
Antineoplásicos/farmacologia , Creatina Quinase/metabolismo , Creatina/farmacologia , Neoplasias Musculares/metabolismo , Músculo Esquelético/metabolismo , Sarcoma/metabolismo , Amidinotransferases , Animais , Ácido Ascórbico/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Creatina/metabolismo , Guanidinoacetato N-Metiltransferase , Humanos , Proteínas de Membrana Transportadoras , Camundongos , Neoplasias Musculares/tratamento farmacológico , Neoplasias Musculares/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Aldeído Pirúvico/farmacologia , Sarcoma/tratamento farmacológico , Sarcoma/patologiaRESUMO
We have purified a novel protein from mice muscle, which through N-terminal amino acid sequencing was identified as a truncated form of mouse albumin. The protein was found to be a monomer of approximately 64 kDa and located in the cytosol. The purified protein strongly crossreacted with commercial albumin antibody. Presence of this protein was observed in different mouse organs. Further biochemical studies as well as CD spectroscopy indicated that the protein binds D-glyceraldehyde-3-phosphate limiting the availability of the substrate to the enzyme D-glyceraldehyde-3-phosphate dehydrogenase, thereby inhibiting its catalytic activity. The implication of this protein in the control of glycolysis has been discussed.
Assuntos
Proteínas de Transporte/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Albuminas/genética , Albuminas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Citosol/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Gliceraldeído 3-Fosfato/genética , Concentração de Íons de Hidrogênio , Camundongos , Dados de Sequência Molecular , Peso Molecular , Músculo Esquelético/enzimologia , Ligação Proteica/genética , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Especificidade por Substrato/genética , Distribuição TecidualRESUMO
In vertebrates, phosphocreatine and ATP are continuously interconverted by the reversible reaction of creatine kinase in accordance with cellular energy needs. Sarcoma tissue and its normal counterpart, creatine-rich skeletal muscle, are good source materials to study the status of creatine and creatine kinase with the progression of malignancy. We experimentally induced sarcoma in mouse leg muscle by injecting either 3-methylcholanthrene or live sarcoma 180 cells into one hind leg. Creatine, phosphocreatine and creatine kinase isoform levels decreased as malignancy progressed and reached very low levels in the final stage of sarcoma development; all these parameters remained unaltered in the unaffected contralateral leg muscle of the same animal. Creatine and creatine kinase levels were also reduced significantly in frank malignant portions of human sarcoma and gastric and colonic adenocarcinoma compared with the distal nonmalignant portions of the same samples. In mice, immunoblotting with antibodies against cytosolic muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase showed that both of these isoforms decreased as malignancy progressed. Expressions of mRNA of muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase were also severely downregulated. In human sarcoma these two isoforms were undetectable also. In human gastric and colonic adenocarcinoma, brain-type creatine kinase was found to be downregulated, whereas ubiquitous mitochondrial creatine kinase was upregulated. These significantly decreased levels of creatine and creatine kinase isoforms in sarcoma suggest that: (a) the genuine muscle phenotype is lost during sarcoma progression, and (b) these parameters may be used as diagnostic marker and prognostic indicator of malignancy in this tissue.
Assuntos
Transformação Celular Neoplásica/metabolismo , Creatina Quinase/metabolismo , Creatina/metabolismo , Músculo Esquelético/metabolismo , Neoplasias de Tecido Muscular/metabolismo , Fosfocreatina/metabolismo , Sarcoma/metabolismo , Adenocarcinoma/enzimologia , Animais , Neoplasias Colorretais/enzimologia , Creatina Quinase/análise , Creatina Quinase/genética , Creatina Quinase Forma BB/análise , Creatina Quinase Forma MM/análise , Creatina Quinase Mitocondrial/análise , Progressão da Doença , Humanos , Immunoblotting , Camundongos , Músculo Esquelético/enzimologia , Neoplasias de Tecido Muscular/enzimologia , Neoplasias de Tecido Muscular/patologia , RNA Mensageiro/metabolismo , Sarcoma/enzimologia , Sarcoma Experimental/enzimologia , Sarcoma Experimental/metabolismo , Sarcoma Experimental/patologia , Neoplasias Gástricas/enzimologiaRESUMO
A historical perspective on methylglyoxal research is briefly presented, mentioning the documented anticancer and antiviral effects of methylglyoxal. The idea and the supporting experimental evidence of Albert Szent-Györgyi et al. that methylglyoxal is a natural growth regulator and can act as an anticancer agent are mentioned. Previously a few in vivo studies suggested safe administration of methylglyoxal. However, recent literature abounds with the toxic effects of methylglyoxal. The authors present a brief critical overview of studies indicating both toxic and beneficial effects of methylglyoxal and suggest that the beneficial effects of methylglyoxal outweigh its toxic effects. Encouraged by the studies of Szent-Györgyi et al., the present authors undertook systematic investigations to understand the mechanism of the anticancer effect of methylglyoxal. The results of these investigations led to the proposal that the fundamental changes in malignant cells are critical alterations of glyceraldehyde-3-phosphate dehydrogenase and mitochondrial complex I, and methylglyoxal's anticancer effect might be mediated by acting on these altered sites. Moreover, a new hypothesis on cancer has been proposed, suggesting that excessive ATP formation in cells may lead to malignancy. Toxicity and pharmacokinetic studies were performed on animals and it was observed that methylglyoxal is potentially safe for humans. A methylglyoxal-based anticancer formulation was developed and a three-phase study of treating a total number of 86 cancer patients was carried out. The results appear to be promising. Most of the cancer patients benefited greatly and a significant number of patients became free of the disease. Contrary to the effect of existing anticancer drugs, this methylglyoxal-based formulation is devoid of any toxic effect and reasonably effective against a wide variety of cancers. The symptomatic improvements of the many patients who died of progressive disease suggest that the formulation could also be used for palliation. The authors urge the scientific community to test the formulation and if found effective then to improve it further.
Assuntos
Antineoplásicos/farmacologia , Aldeído Pirúvico/farmacologia , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/uso terapêutico , Ensaios Clínicos como Assunto , Complicações do Diabetes/metabolismo , Feminino , Produtos Finais de Glicação Avançada/biossíntese , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Aldeído Pirúvico/metabolismo , Aldeído Pirúvico/uso terapêuticoRESUMO
The surface exponents, scaling behavior, and bulk porosity of a generalized ballistic deposition (GBD) model are studied. In nature, there exist particles with varying degrees of stickiness ranging from completely nonsticky to fully sticky. Such particles may adhere to any one of the successively encountered surfaces, depending on a sticking probability that is governed by the underlying stochastic mechanism. The microscopic configurations possible in this model are much larger than those allowed in existing models of ballistic deposition and competitive growth models that seek to mix ballistic and random deposition processes. In this article, we find the scaling exponents for surface width and porosity for the proposed GBD model. In terms of scaled width W[over Ì] and scaled time t[over Ì], the numerical data collapse onto a single curve, demonstrating successful scaling with sticking probability p and system size L. Similar scaling behavior is also found for the porosity.
RESUMO
PURPOSE: The normal metabolite methylglyoxal (MG) specifically kills cancer cells by inhibiting glycolysis and mitochondrial respiration without much adverse effect upon normal cells. Though the anticancer property of MG is well documented, its gradual enzymatic degradation in vivo has prompted interest in developing a nanoparticulate drug delivery system to protect it and also to enhance its efficacy. MATERIALS AND METHODS: MG-conjugated chitosan nanoparticles (Nano-MG) were prepared by conjugating the carbonyl group of MG with the amino group of chitosan polymer (Schiff's base formation). Nano-MG were characterized in detail using the dynamic light scattering method, zeta potential measurement, Fourier transform infrared spectroscopy, and transmission electron microscopic analysis. Amount of MG anchored to Nano-MG, stability of Nano-MG, and in vitro release of MG from Nano-MG were estimated spectrophotometrically. Ehrlich ascites carcinoma (EAC) cells, human breast cancer cell line HBL-100, and lung epithelial adenocarcinoma cell line A549 were used as test systems to compare Nano-MG with bare MG in vitro. Cytotoxicity to EAC cells was evaluated by the trypan blue dye exclusion test, and cell viability of HBL-100 and A549 cells were studied using 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis of HBL-100 cells was assessed by flow cytometry and confocal microscopy. In vivo studies were performed on both EAC cells inoculated and also in sarcoma-180-induced solid tumor-bearing Swiss albino mice to assess the anticancer activity of Nano-MG in comparison to bare MG with varying doses, times, and administrative routes. RESULTS: Fourier transform infrared spectroscopy revealed the presence of imine groups in Nano-MG due to conjugation of the amino group of chitosan and carbonyl group of MG with diameters of nanoparticles ranging from 50-100 nm. The zeta potential of Nano-MG was +21 mV and they contained approximately 100 µg of MG in 1 mL of solution. In vitro studies with Nano-MG showed higher cytotoxicity and enhanced rate of apoptosis in the HBL-100 cell line in comparison with bare MG, but no detrimental effect on normal mouse myoblast cell line C2C12 at the concerned doses. Studies with EAC cells also showed increased cell death of nearly 1.5 times. Nano-MG had similar cytotoxic effects on A549 cells. In vivo studies further demonstrated the efficacy of Nano-MG over bare MG and found them to be about 400 times more potent in EAC-bearing mice and nearly 80 times more effective in sarcoma-180-bearing mice. Administration of ascorbic acid and creatine during in vivo treatments augmented the anticancer effect of Nano-MG. CONCLUSION: The results clearly indicate that Nano-MG may constitute a promising tool in anticancer therapeutics in the near future.
Assuntos
Antineoplásicos , Quitosana , Nanopartículas , Aldeído Pirúvico , Animais , Antineoplásicos/química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quitosana/química , Quitosana/toxicidade , Humanos , Camundongos , Nanopartículas/química , Nanopartículas/toxicidade , Nanotecnologia , Aldeído Pirúvico/química , Aldeído Pirúvico/toxicidadeRESUMO
Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (GA3PD) (EC. 1.2.1.12) was completely inactivated by diethyl pyrocarbonate (DEPC), a fairly specific reagent for histidine residues in the pH range of 6.0-7.5. The rate of inactivation was dependent on pH and followed pseudo-first order reaction kinetics. The difference spectrum of the inactivated and native enzymes showed an increase in the absorption maximum at 242 nm, indicating the modification of histidine residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of one essential histidine residue to be responsible for loss of the catalytic activity of EAC cell GA3PD. DEPC inactivation was protected by substrates, D-glyceraldehyde-3-phosphate and NAD, indicating the presence of essential histidine residue at the substrate-binding region of the active site. Double inhibition studies also provide evidence for the presence of histidine residue at the active site.
Assuntos
Carcinoma de Ehrlich/metabolismo , Regulação Enzimológica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/biossíntese , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Histidina/química , Animais , Domínio Catalítico , Dietil Pirocarbonato/química , Ácido Ditionitrobenzoico/química , Concentração de Íons de Hidrogênio , Hidroxilamina/química , Cinética , CamundongosRESUMO
3-Phosphoglycerate kinase (3-PGK) has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells by (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. The enzyme has been partially characterized and compared with the characteristics of this enzyme of other normal and malignant cells. The EAC cell 3-PGK is composed of a single subunit of 47 kDa. It has a broad pH optimum (pH 6.0-7.5) for its enzymatic activity. The apparent Km values of 3-phosphoglycerate (3-PGA) and ATP for 3-PGK have been found out to be 0.25 mM and 0.1 mM respectively. Similar to 3-PGK of other cells, the EAC enzyme requires either Mg2+ or Mn2+ for full activity; the optimum concentrations of Mg2+ and Mn2+ are 0.8 mM and 0.5 mM respectively. When ATP and 3-PGA act as substrates, ADP, the reaction product of 3-PGK-catalyzed reaction has been found to inhibit this enzyme. Kinetic studies were made on the inhibition of ADP in presence of the substrates ATP and 3-PGA. Attempts to hybridize 3-PGK and glyceraldehyde-3-phosphate dehydrogenase of EAC cells by NAD or glutaraldehyde were unsuccessful.
Assuntos
Carcinoma de Ehrlich/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fosfoglicerato Quinase/química , Fosfoglicerato Quinase/isolamento & purificação , Difosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica/métodos , Ácido Edético/química , Concentração de Íons de Hidrogênio , Cinética , Magnésio/química , Manganês/química , Mercaptoetanol/farmacologia , Camundongos , Transplante de Neoplasias , Hibridização de Ácido NucleicoRESUMO
The surface and bulk properties of a modified ballistic deposition model are investigated. The deposition rule interpolates between nearest- and next-nearest-neighbor ballistic deposition and the random deposition models. The stickiness of the depositing particle is controlled by a parameter and the type of interparticle force. Two such forces are considered: Coulomb and van der Waals type. The interface width shows three distinct growth regions before eventual saturation. The rate of growth depends more strongly on the stickiness parameter than on the type of interparticle force. However, the porosity of the deposits is strongly influenced by the interparticle force.
Assuntos
Modelos Teóricos , Propriedades de Superfície , Simulação por Computador , PorosidadeRESUMO
The creatine/creatine kinase system decreases drastically in sarcoma. In the present study, an investigation of catalytic activities, western blot and mRNA expression unambiguously demonstrates the prominent expression of the creatine-synthesizing enzymes l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase in sarcoma, Ehrlich ascites carcinoma and Sarcoma 180 cells, whereas both enzymes were virtually undetectable in normal muscle. Compared to that of normal animals, these enzymes remained unaffected in the kidney or liver of sarcoma-bearing mice. High activity and expression of mitochondrial arginase II in sarcoma indicated increased ornithine formation. Slightly or moderately higher levels of ornithine, guanidinoacetate and creatinine were observed in sarcoma compared to muscle. Despite the intrinsically low level of creatine in Ehrlich ascites carcinoma and Sarcoma 180 cells, these cells could significantly take up and release creatine, suggesting a functional creatine transport, as verified by measuring mRNA levels of creatine transporter. Transcript levels of arginase II, ornithine-decarboxylase, S-adenosyl-homocysteine hydrolase and methionine-synthase were significantly upregulated in sarcoma and in Ehrlich ascites carcinoma and Sarcoma 180 cells. Overall, the enzymes related to creatine and arginine/methionine metabolism were found to be significantly upregulated in malignant cells. However, the low levels of creatine kinase in the same malignant cells do not appear to be sufficient for the building up of an effective creatine/phosphocreatine pool. Instead of supporting creatine biosynthesis, l-arginine:glycine amidinotransferase and N-guanidinoacetate methyltransferase appear to be geared to support cancer cell metabolism in the direction of polyamine and methionine synthesis because both these compounds are in high demand in proliferating cancer cells.
Assuntos
Arginina/metabolismo , Creatina/biossíntese , Enzimas/metabolismo , Metionina/metabolismo , Neoplasias/enzimologia , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Adenosil-Homocisteinase/genética , Amidinotransferases/genética , Amidinotransferases/metabolismo , Animais , Arginase/genética , Arginase/metabolismo , Carcinoma de Ehrlich/enzimologia , Carcinoma de Ehrlich/metabolismo , Carcinoma de Ehrlich/patologia , Linhagem Celular Tumoral , Creatina/sangue , Creatina/metabolismo , Creatinina/sangue , Creatinina/metabolismo , Enzimas/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Guanidinoacetato N-Metiltransferase/genética , Guanidinoacetato N-Metiltransferase/metabolismo , Rim/enzimologia , Rim/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Proteínas de Membrana Transportadoras/genética , Camundongos , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Ornitina/sangue , Ornitina/metabolismo , Ornitina Descarboxilase/genética , Sarcoma 180/enzimologia , Sarcoma 180/metabolismo , Sarcoma 180/patologiaRESUMO
Previous in vivo studies from several laboratories had shown remarkable curative effect of methylglyoxal on cancer-bearing animals. In contrast, most of the recent in vitro studies have assigned a toxic role for methylglyoxal. The present study was initiated with the objective to resolve whether methylglyoxal is truly toxic in vivo and to reassess its therapeutic potential. Four species of animals, both rodent and non-rodent, were treated with different doses of methylglyoxal through oral, subcutaneous and intravenous routes. Acute (treatment for only 1 day) toxicity tests had been done with mouse and rat. These animals received 2, 1 and 0.3 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. Chronic (treatment for around a month) toxicity test had been done with mouse, rat, rabbit and dog. Mouse, rat and dog received 1, 0.3 and 0.1 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. Rabbit received 0.55, 0.3 and 0.1 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. It had been observed that methylglyoxal had no deleterious effect on the physical and behavioral pattern of the treated animals. Fertility and teratogenecity studies were done with rats that were subjected to chronic toxicity tests. It had been observed that these animals produced healthy litters indicating no damage of the reproductive systems as well as no deleterious effect on the offspring. Studies on several biochemical and hematological parameters of methylglyoxal-treated rats and dogs and histological studies of several organs of methylglyoxal-treated mouse were performed. These studies indicated that methylglyoxal had no apparent deleterious effect on some vital organs of these animals. A detailed pharmacokinetic study was done with mouse after oral administration of methylglyoxal. The effect of methylglyoxal alone and in combination with creatine and ascorbic acid on cancer-bearing animals had been investigated by measuring the increase in life span and tumor cell growth inhibition. The results indicated that anticancer effect of methylglyoxal was significantly augmented by ascorbic acid and further augmented by ascorbic acid and creatine. Nearly 80% of the animals treated with methylglyoxal plus ascorbic acid plus creatine were completely cured and devoid of any malignant cells within the peritoneal cavity.
Assuntos
Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Ácido Ascórbico/uso terapêutico , Carcinoma de Ehrlich/tratamento farmacológico , Creatina/uso terapêutico , Aldeído Pirúvico/farmacocinética , Aldeído Pirúvico/toxicidade , Vitaminas/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Peso Corporal/efeitos dos fármacos , Carcinoma de Ehrlich/patologia , Cães , Sinergismo Farmacológico , Enzimas/sangue , Feminino , Fertilidade/efeitos dos fármacos , Longevidade/efeitos dos fármacos , Masculino , Camundongos , Transplante de Neoplasias , Aldeído Pirúvico/uso terapêutico , Coelhos , Ratos , Reprodução/efeitos dos fármacos , Especificidade da Espécie , Análise de Sobrevida , Teratogênicos/toxicidadeRESUMO
We had previously shown that creatine exerted a protective effect against inhibition of cardiac mitochondrial respiration by methylglyoxal (SinhaRoy S, Biswas S, Ray M, Ray S. Biochem J 372: 661-669,2003). In the present study, we have investigated the mechanism of this protective effect by specific amino acid modifying reagent and by several compounds, which are structurally related to creatine. The results show that the compounds, which contain guanidine group such as arginine and guanidinopropionic acid, exert a protective effect, which is quantitatively similar to creatine. This result suggests the presence of carboxylic acid(s) such as glutamic and/or aspartic acid(s) in the creatine-binding site, which has been further supported by experiments with N-ethyl-5-phenyl isoxazolium-3'-sulfonate a reagent known to modify these amino acids. Both polarographic and spectrophotometric assays were performed with NADH as respiratory substrate by using a) submitochondrial particles by sonication, b) freeze-thawed mitochondria and c) mitochondria permeabilized by alamethicin treatment. The results of these studies as compared to that of intact mitochondria indicate that structural integrity of mitochondria is essential for the protective effect of creatine.
Assuntos
Ácido Aspártico/metabolismo , Creatina/farmacologia , Ácido Glutâmico/metabolismo , Mitocôndrias Cardíacas/metabolismo , Substâncias Protetoras/farmacologia , Alameticina/farmacologia , Animais , Ácido Aspártico/química , Sítios de Ligação , Respiração Celular/efeitos dos fármacos , Creatina/análogos & derivados , Complexo I de Transporte de Elétrons/efeitos dos fármacos , Complexo I de Transporte de Elétrons/metabolismo , Ácido Glutâmico/química , Cabras , Membranas Intracelulares/efeitos dos fármacos , Isoxazóis/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , NAD/metabolismo , NADH Desidrogenase/efeitos dos fármacos , NADH Desidrogenase/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Aldeído Pirúvico/efeitos adversos , Partículas Submitocôndricas/efeitos dos fármacos , Partículas Submitocôndricas/metabolismoRESUMO
Previous publications from our laboratory have shown that methylglyoxal inhibits mitochondrial respiration of malignant and cardiac cells, but it has no effect on mitochondrial respiration of other normal cells [Biswas, Ray, Misra, Dutta and Ray (1997) Biochem. J. 323, 343-348; Ray, Biswas and Ray (1997) Mol. Cell. Biochem. 171, 95-103]. However, this inhibitory effect of methylglyoxal is not significant in cardiac tissue slices. Moreover, post-mitochondrial supernatant (PMS) of cardiac cells could almost completely protect the mitochondrial respiration against the inhibitory effect of methylglyoxal. A systematic search indicated that creatine present in cardiac cells is responsible for this protective effect. Glutathione has also some protective effect. However, creatine phosphate, creatinine, urea, glutathione disulphide and beta-mercaptoethanol have no protective effect. The inhibitory and protective effects of methylglyoxal and creatine respectively on cardiac mitochondrial respiration were studied with various concentrations of both methylglyoxal and creatine. Interestingly, neither creatine nor glutathione have any protective effect on the inhibition by methylglyoxal on the mitochondrial respiration of Ehrlich ascites carcinoma cells. The creatine and glutathione contents of several PMS, which were tested for the possible protective effect, were measured. The activities of two important enzymes, namely glyoxalase I and creatine kinase, which act upon glutathione plus methylglyoxal and creatine respectively, were also measured in different PMS. Whether mitochondrial creatine kinase had any role in the protective effect of creatine had also been investigated using 1-fluoro-2,4-dinitrobenzene, an inhibitor of creatine kinase. The differential effect of creatine on mitochondria of cardiac and malignant cells has been discussed with reference to the therapeutic potential of methylglyoxal.