RESUMEN
Gamma-aminobutyric acid (GABA) is considered to be a multifunctional molecule with various physiological effects throughout the body. It is also evident that the liver contains GABA and its transporter. However, the functions of GABA in liver have not been well documented. In this study, the cytoprotective effect of GABA against ethanol-induced hepatotoxicity was evaluated in primary cultured rat hepatocytes. Addition of ethanol induced decrease of cell viability in a dose-dependent manner. However, treatment with GABA resulted in a dose-dependent recovery from ethanol (150 mM)-induced cytotoxicity.GABA reversed the ethanol-induced decrease in intracellular polyamine levels. Furthermore, the addition of polyamines also reversed the ethanol-induced decrease of cell viability. These results suggest that GABA is protective against the cytotoxicity of ethanol in isolated rat hepatocytes and this effect may be modulated by the maintenance of intracellular polyamine levels.
Asunto(s)
Poliaminas Biogénicas/metabolismo , Citotoxinas/toxicidad , Etanol/toxicidad , Hepatocitos/metabolismo , Ácido gamma-Aminobutírico/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Hepatocitos/patología , Masculino , Ratas , Ratas WistarRESUMEN
1'-Acetoxychavicol acetate (ACA) has been shown to inhibit tumor cell growth, but there is limited information on its effects on cell signaling and the cell cycle control pathway. In this study, we sought to determine how ACA alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells (EATC). ACA caused an accumulation of cells in the G1 phase and an inhibition of DNA synthesis, which were reversed by supplementation with N-acetylcysteine (NAC) or glutathione ethyl ester (GEE). Furthermore, ACA decreased hyperphosphorylated Rb levels and increased hypophosphorylated Rb levels. NAC and GEE also abolished the decease in Rb phosphorylation by ACA. As Rb phosphorylation is regulated by G1 cyclin dependent kinase and CDK inhibitor p27(kip1), which is an important regulator of the mammalian cell cycle, we estimated the amount of p27(kip1) levels by western blotting. Treatment with ACA had virtually no effect on the amount of p27(kip1) levels, but caused a decrease in phosphorylated p27(kip1) and an increase in unphosphorylated p27(kip1) as well as an increase in the nuclear localization of p27(kip1). These events were abolished in the presence of NAC or GEE. These results suggest that in EATC, cell growth inhibition elicited by ACA involves decreases in Rb and p27(kip1) phosphorylation and an increase in nuclear localization of p27(kip1), and these events are dependent on the cellular thiol status.
Asunto(s)
Ciclo Celular/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteína de Retinoblastoma/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Terpenos/metabolismo , Acetilcisteína/metabolismo , Animales , Alcoholes Bencílicos , Carcinoma de Ehrlich , Línea Celular Tumoral , ADN/biosíntesis , Glutatión/análogos & derivados , Glutatión/metabolismo , Humanos , Fosforilación , Extractos Vegetales/química , Extractos Vegetales/metabolismo , Compuestos de Sulfhidrilo/química , Terpenos/químicaRESUMEN
We previously demonstrated that evening primrose extract (EPE) induced apoptosis and inhibited the DNA synthesis in Ehrlich ascites tumor cells (EATC) and suggested that EPE-induced inhibition of the growth of EATC are via at least two pathway differentially modulated by reactive oxygen species, notably intracellular peroxides. These are (a) the EPE-induced apoptosis pathway which is dependent on increases in hydrogen peroxide and (b) the EPE-induced inhibition of cell proliferation which is hydrogen peroxide independent. In this study, EPE brought about a significant decrease in intracellular polyamine levels. Furthermore, the addition of polyamines reversed the EPE-induced decrease in cell viability and suppressed the EPE-induced increase in intracellular hydrogen peroxides. However, the addition of polyamines did not reverse EPE-induced decrease in DNA synthesis and phosphorylation of Rb protein, and EPE-induced translocation of AIF. These results suggest the involvement of polyamines in the EPE-induced apoptosis pathway which is dependent on increase in hydrogen peroxide.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma de Ehrlich/patología , Oenothera biennis/química , Extractos Vegetales/farmacología , Poliaminas/farmacología , Animales , Carcinoma de Ehrlich/tratamiento farmacológico , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión/métodos , ADN de Neoplasias/biosíntesis , ADN de Neoplasias/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Extractos Vegetales/química , Poliaminas/análisis , Poliaminas/química , Putrescina/farmacología , Espermidina/farmacología , Espermina/farmacología , Células Tumorales CultivadasRESUMEN
The purpose of this study was to investigate the possible molecular mechanisms of the antiproliferative effect induced by protocatechualdehyde (PA, a dihydroxybenzene derivative). The viability of cytotoxic T cells (CfLL-2) stimulated by IL2 was significantly inhibited at 0.12 mM PA. This inhibitory effect was associated with the induction of apoptosis detected by DNA fragmentation assay. DNA ladder appeared at 0.12 mM PA and the intensity of DNA ladder was visible at 0.3 mM PA. PA inhibited the Ib2-dependent tyrosine phosphorylation of 91, 80 and 55 KDa proteins, but did not affect IL2-dependent serine/threonine phosphorylation of proteins. The levels of bcl-2 protein and mRNA were suppressed by PA. An alteration in bax protein expression on the apoptosis process in CTLL-2 cells was not observed. However, caspase-3 activity was increased by PA. Our results demonstrate that PA inhibited cell proliferation and induced apoptosis in CTLL-2 cells. It is concluded that PA is a potent anti-proliferative agent and is expected to be a promising candidate for novel therapeutics.
Asunto(s)
Apoptosis , Benzaldehídos/farmacología , Catecoles/farmacología , Inhibidores de Crecimiento/farmacología , Caspasa 3 , Caspasas/biosíntesis , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Fosforilación , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/metabolismo , Proteína X Asociada a bcl-2RESUMEN
We reported previously that the mechanism by which Green tea extract (GTE) elicited growth-inhibitory effects in Ehrlich ascites tumor cells involved a decrease in ornithine decarboxylase (ODC) activity and in cell viability. Decrease in ODC activity has been associated with apoptotic cell death and we therefore studied changes in cytochrome c release and caspase activation, which characterize apoptosis. GTE caused a dose- and time-dependent increase in caspase-3-like protease activation, preceded by a release of cytochrome c from the mitochondria. Inhibiting the activation of caspase-3 with acetyl-Asp-Glu-Val-Asp-alpha-aldehyde (caspase inhibitor) caused a reversal in the effect on cell viability.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma de Ehrlich/metabolismo , Carcinoma de Ehrlich/patología , Caspasas/metabolismo , Grupo Citocromo c/metabolismo , Té/química , Animales , Caspasa 3 , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática , Cinética , Ratones , Oligopéptidos/farmacología , Extractos Vegetales/farmacologíaRESUMEN
The effect of green tea extract (GTE) in Ehrlich ascites tumor cells (EATC) was studied with respect to changes in the intracellular kinase system involving mitogen-activated protein kinases (MAPKs) and cellular thiol. We have previously shown a reduction in viability of EATC and tyrosine phosphorylation of 42 and 45 kDa proteins by GTE and its polyphenolic component, Epigallocatechin (EGC) (D.O. Kennedy, S. Nishimura, T. Hasuma, Y. Yoshihisa, S. Otani, I. Matsui-Yuasa, Involvement of protein tyrosine phosphorylation in the effect of green tea polyphenols on Ehrlich ascites tumor cells in vitro, Chem. Biol. Interact. 110 (1998) 159-172). Furthermore, GTE and EGC significantly decreased both cellular non-protein and protein sulfhydryl levels in EATC, but replenishing thiol stores with N-acetylcysteine (NAC) caused a recovery in cell viability, and therefore SH groups were identified as a novel target of green tea cytotoxicity (D.O. Kennedy, M. Matsumoto, A. Kojima, I. Matsui-Yuasa, Cellular thiol status and cell death in the effect of green tea polyphenols in Ehrlich ascites tumor cells, Chem. Biol. Interact. 122 (1999) 59-71). In this study, we have observed the stimulation of three forms of MAPK, namely ERK1/2, JNK/SAPK and p38, by EGC, which were dose and time-dependent. These MAPK stimulations were found to be cellular thiol status-dependent events as NAC reversed these stimulations. Furthermore, inhibition of the p38 MAPK pathway using the p38 inhibitor SB203580 caused a marked dose-dependent reduction in the decrease in cell viability caused by EGC treatment. Inhibiting the Erk1/2 MAPK pathway using the MEK inhibitor PD098059 caused a slight change in the decrease in cell viability by EGC. These may suggest that the cytotoxicity associated with EGC was more associated with the other MAPKs than with ERK1/2. This may be the first study of its kind providing a novel evidence of a role for different forms of MAPKs in the antitumor effect of green tea polyphenols, especially EGC, in Ehrlich ascites tumor cells.
Asunto(s)
Carcinoma de Ehrlich/tratamiento farmacológico , Catequina/análogos & derivados , Flavonoides/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fitoterapia , Té/uso terapéutico , Acetilcisteína/farmacología , Animales , Antineoplásicos/farmacología , Carcinoma de Ehrlich/metabolismo , Carcinoma de Ehrlich/patología , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Cinética , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fenoles/farmacología , Extractos Vegetales/farmacología , Polímeros/farmacología , Compuestos de Sulfhidrilo/metabolismo , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The relationship between cellular glutathione (GSH), protein-SH levels, and lactate dehydrogenase (LDH), with respect to the effect of polyamines on the cytoprotective ability of L-cysteine and L-methionine, the most important components in the sulfur amino acid metabolic pathway, in carbon tetrachloride (CCl4)-induced toxicity in isolated rat hepatocytes was studied. CCl4 induced a LDH release and decreased cellular thiols and polyamines levels but treatment with L-cysteine and L-methionine reversed these decreases. Treating with methylglyoxal bis-(guanylhydrazone), MGBG, an irreversible inhibitor of S-adenosylmethionine decarboxylase, which is a key enzyme in spermidine and spermine biosynthesis, and therefore used to deplete cellular polyamines, prevented the protective effect of L-cysteine and L-methionine, but the addition of exogenous polyamines inhibited the influence of MGBG. These results suggest that the cytoprotective effect of L-cysteine and L-methionine in CCl4-induced toxicity were via maintenance of cellular polyamines, GSH and protein-SH concentrations and prevention of LDH leakage.
Asunto(s)
Tetracloruro de Carbono/toxicidad , Cisteína/farmacología , Citoprotección/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Metionina/farmacología , Poliaminas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Glutatión/metabolismo , Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Masculino , Maleatos/farmacología , Mitoguazona/farmacología , Ratas , Ratas Sprague-Dawley , S-Adenosilmetionina/antagonistas & inhibidoresRESUMEN
OBJECTIVE: To investigate polyamine metabolism in rheumatoid synovial adherent cells stimulated by interleukin- 1beta (IL-1beta). METHODS: Synovial adherent cells obtained from patients with rheumatoid arthritis (RA) were cultured and incubated in the presence or absence of human recombinant IL-1beta at a concentration of 10 ng/ml for 24 h. The cellular contents of polyamines as well as the activities of spermidine/spermine N1-acetyltransferase (SAT) and ornithine decarboxylase (ODC) were measured. RESULTS: Polyamines in synovial adherent cells decreased significantly after 24 h incubation in the absence of IL-1beta. However, in the presence of IL-Ibeta, putrescine and N'-acetylspermidine increased significantly. No significant difference was observed between the amount of spermidine in synovial adherent cells incubated with and without IL-1beta. Spermine and N8-acetylspermidine in synovial adherent cells incubated with IL-1beta decreased significantly more than in synovial adherent cells incubated without. SAT activity reached a peak 12 h after the addition of IL-1beta and then decreased, while the ODC activity did not increase. SAT activity was elevated by the addition of IL-1beta in a dose dependent manner. CONCLUSION: An increase in the putrescine level in rheumatoid synovial adherent cells as a result of the elevation of SAT activity induced by IL-1beta may play a role in RA.
Asunto(s)
Acetiltransferasas/metabolismo , Artritis Reumatoide/metabolismo , Interleucina-1/farmacología , Putrescina/metabolismo , Membrana Sinovial/enzimología , Artritis Reumatoide/inmunología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Colagenasas/metabolismo , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Expresión Génica/fisiología , Humanos , Metaloproteinasa 3 de la Matriz/genética , Espermidina/metabolismo , Espermina/metabolismo , Membrana Sinovial/citología , Membrana Sinovial/efectos de los fármacosRESUMEN
Zn(2+) has multiple implications in cellular metabolism, including free radicals metabolism and cell death by apoptosis. In the present study, we examined the role of Zn(2+) in the regulation of apoptosis in cultured rat hepatocytes. The chelation of Zn(2+) by a membrane permeable metal ion chelator, N, N, N', N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), induced apoptosis. Addition of ZnSO(4) prevented TPEN-induced apoptosis. Unlike the effect of TPEN, a membrane impermeable metal ion chelator, diethylenetriamine pentaacetic acid (DTPA), did not induce apoptosis, indicating that chelation of intracellular Zn(2+) was required to trigger apoptosis. Caspase-3-like proteolytic activity, a general biochemical mediator of apoptosis in a variety of cells and tissues, was also activated with the treatment of TPEN but not DTPA. TPEN treatment, but not DTPA, also resulted in the depletion of intracellular reduced glutathione (GSH) but addition of Zn(2+) recovered the GSH level. N-acetyl-L-cysteine (NAC), a thiol antioxidant, prevented TPEN-induced apoptosis. These results taken together suggest that intracellular Zn(2+) interfere with the apoptosis process, possibly through the regulation of cellular redox potential involving GSH.
Asunto(s)
Apoptosis , Quelantes/farmacología , Glutatión/metabolismo , Hígado/citología , Zinc/metabolismo , Acetilcisteína/farmacología , Animales , Caspasa 3 , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Interacciones Farmacológicas , Etilenodiaminas/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The efficacy of the antitumor activity of 1'-acetoxychavicol acetate (ACA), reported to be a suppressor of chemically induced carcinogenesis, was evaluated in Ehrlich ascites tumor cells. ACA treatment resulted in changes in morphology and a dose-dependent suppression of cell viability. Apoptosis, characterized by nuclear condensation, membrane blebbing, cell shrinkage and a significant induction of caspase-3-like protease activity at 8 h in a time-course study were observed. Formation of apoptotic bodies was preceded by lowering of intracellular polyamines, particularly putrescine, and both dose- and time-dependent inhibitory and activation effect by ACA on ornithine decarboxylase (ODC) and spermidine/spermine N(1)-acetyltransferase (SSAT), respectively. Administration of exogenous polyamines prevented ACA-induced apoptosis represented by a reduction in the number of apoptotic bodies and also caused reduction in the induced caspase-3-like protease activity at 8 h. These findings suggest that the anticarcinogenic effects of ACA might be partly due to perturbation of the polyamine metabolic pathway and triggering of caspase-3-like activity, which result in apoptosis.
Asunto(s)
Anticarcinógenos/toxicidad , Apoptosis/efectos de los fármacos , Carcinoma de Ehrlich/metabolismo , Caspasas/metabolismo , Poliaminas/metabolismo , Terpenos/toxicidad , Acetiltransferasas/metabolismo , Animales , Alcoholes Bencílicos , Carcinoma de Ehrlich/patología , Caspasa 3 , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Supervivencia Celular/efectos de los fármacos , Activación Enzimática , Cinética , Ratones , Ornitina Descarboxilasa/metabolismo , Células Tumorales CultivadasRESUMEN
Epidemiological studies suggest that the consumption of green tea may help prevent cancers in humans, and also breast and prostate cancers in animal models are reduced by green tea, and several mechanisms have been proposed for these effects. In this study the relationship between cellular sulfhydryl (SH) groups and the cytotoxicity of green tea polyphenols in Ehrlich ascites tumor cells was examined. It was found that in the presence of green tea extract (GTE) (100 microg/ml) and one of its polyphenolic components, epigallocatechin (EGC; 100 microM), both cellular non-protein (GSH) and protein-sulfhydryl (PSH) levels were significantly decreased and this was associated with a decrease in cell viability. Replenishing the thiol levels by using N-acetylcysteine (NAC) caused a recovery in cell viability, but this recovery was dependent on the time of thiol replenishment in the presence of EGC (initial 15 min). These results identify SH groups as a novel target of green tea polyphenols cytotoxicity in tumor cells, and a regulatory role for green tea in terms of reducing sulfhydryls in tumor inhibition.
Asunto(s)
Carcinoma de Ehrlich/metabolismo , Muerte Celular/efectos de los fármacos , Flavonoides , Fenoles/farmacología , Extractos Vegetales/farmacología , Polímeros/farmacología , Compuestos de Sulfhidrilo/metabolismo , Té , Acetilcisteína/farmacología , Animales , Carcinoma de Ehrlich/patología , Supervivencia Celular/efectos de los fármacos , Glutatión/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Estructura Molecular , Polifenoles , Células Tumorales CultivadasRESUMEN
BACKGROUND/AIMS: Hepatocyte growth factor and transforming growth factor-alpha are growth factors with important roles in hepatocyte proliferation. The polyamines, putrescine, spermidine, and spermine are widely distributed in many different cells and play an essential role in cell growth and differentiation. The present study examined the role of polyamine in this growth promoting factor-induced hepatocyte proliferation, in primary cultured rat hepatocytes. METHODOLOGY: Hepatocytes were isolated from rats by the collagenase perfusion method. Ornithine decarboxylase and S-adenosylmethionine decarboxylase activities were measured as the release of 14CO2 from L-[-14C]ornithine and S-adenosyl-L-[carboxyl14C]methionine, respectively. The concentration of polyamine was analyzed by high performance liquid chromatography. RESULTS: When transforming growth factor-alpha and hepatocyte growth factor were added to the hepatocyte culture simultaneously, ornithine decarboxylase activity, S-adenosylmethionine decarboxylase activity, polyamine concentration and DNA synthesis increased additively. The increase in DNA synthesis caused by transforming growth factor-alpha, hepatocyte growth factor, or both was completely inhibited by alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone). The inhibition was reversed by exogenous spermidine or spermine, but not by putrescine. CONCLUSIONS: Increased spermidine or spermine levels are essential for hepatocyte proliferation in cultured rat hepatocytes.
Asunto(s)
División Celular/fisiología , Replicación del ADN/fisiología , Factor de Crecimiento de Hepatocito/fisiología , Hígado/citología , Poliaminas/metabolismo , Factor de Crecimiento Transformador alfa/fisiología , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Masculino , Ratas , Ratas WistarRESUMEN
We examined the expression of smooth muscle cytoskeleton in spindle-shaped cells in the capsule of hepatocellular carcinoma (HCC) and the septa of liver cirrhosis (LC). Serial sections of livers resected from 11 patients were stained with monoclonal antibodies against vimentin, desmin, smooth muscle actin (1A4, HHF35, CGA7) and smooth muscle myosin heavy chain isoforms (SM1, SM2). Capsular spindle-shaped cells exhibited a cytoskeletal feature indicative of intermediately differentiated smooth muscle cells. Computer-assisted morphometry revealed that the proportions of 1A4-, HHF35-, CGA7- and SM1- positive areas to vimentin-positive area were 88.0+/-11.0%, 50.8+/-17.4%, 25.3+/-16.4% and 19.4+/-12.4% (n=11) in main tumours and 86.6+/-9.4%, 50.9+/-18.7%, 21.1+/-12.3% and 17.6+/-9.7% (n=12) in daughter tumours, indicating that spindle-shaped cells are heterogeneous in cytoskeletal expression. Septal spindle-shaped cells in LC lacked the cytoskeletal proteins specific to differentiated smooth muscle cells (CGA7, SM1, SM2 and desmin). Electron microscopically, capsular spindle-shaped cells contained more microfilaments and less rough endoplasmic reticulum than do septal cells. Intermediately differentiated smooth muscle cells are induced in the capsule of HCC but not in the septa of LC, suggesting a role for stromal interaction by tumour cells in the induction of smooth muscle cells.
Asunto(s)
Carcinoma Hepatocelular/patología , Cirrosis Hepática/patología , Neoplasias Hepáticas/patología , Músculo Liso/patología , Actinas/análisis , Actinas/inmunología , Adulto , Anciano , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/ultraestructura , Diferenciación Celular , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Hígado/química , Hígado/citología , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/química , Neoplasias Hepáticas/ultraestructura , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Músculo Liso/química , Músculo Liso/ultraestructura , Cadenas Pesadas de Miosina/análisis , Cadenas Pesadas de Miosina/inmunología , Isoformas de Proteínas/análisis , Isoformas de Proteínas/inmunologíaRESUMEN
The mechanism by which taurine (2-aminoethanesulfonic acid) protects hepatocytes injury induced by carbon tetrachloride (CCl4) is not fully understood. In a previous study, we reported that cellular polyamines play an important role in this mechanism. The relationship between cellular glutathione (GSH), protein-SH levels, and lactate dehydrogenase (LDH), with respect to the effect of polyamine on the cytoprotective ability of taurine in CCl4-induced toxicity in isolated rat hepatocytes, was examined. CCl4 induced a LDH release and decreased cellular thiols and polyamine levels. Treating with taurine reversed these depletions. The effect of CCl4 was also reversed by the addition of exogenous polyamines. Pretreating with alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, which is a key enzyme in polyamine biosynthesis and therefore used to deplete cellular polyamine, prevented the protective effect of taurine. Adding diethyl maleate, a cellular glutathione-depleting agent, reduced the effect of exogenous polyamines. The role of polyamine in the cytoprotective effect of taurine in CCl4-induced toxicity may therefore be by preventing, among others, GSH and protein-SH depletions.
Asunto(s)
Tetracloruro de Carbono/toxicidad , Hígado/efectos de los fármacos , Poliaminas/farmacología , Compuestos de Sulfhidrilo/farmacología , Taurina/farmacología , Animales , Células Cultivadas , Eflornitina/farmacología , Inhibidores Enzimáticos/farmacología , Hígado/citología , Masculino , Maleatos/farmacología , Inhibidores de la Ornitina Descarboxilasa , Ratas , Ratas Sprague-DawleyRESUMEN
Hepatocyte growth factor (HGF) increased both levels of phosphorylated and non-phosphorylated forms of retinoblastoma protein (RB) in primary cultured rat hepatocytes. Combined treatment of HGF and a specific inhibitor of ornithine decarboxylase (ODC), alpha-difluoromethylornithine (DFMO), reduced the levels of hyper-phosphorylated and hypo-phosphorylated forms of RB and increased the levels of the non-phosphorylated form, compared to HGF alone, but did not affect the total level of RB. Polyamines added exogenously overcame the effects of DFMO; they increased hyper- and hypo-phosphorylated forms and decreased non-phosphorylated RB. TGF-beta1 inhibited the increases in ODC activity, RB phosphorylation, and DNA synthesis induced by HGF. However, polyamines added exogenously could not overcome the inhibition by RB phosphorylation and DNA synthesis by TGF-beta1. These results suggest that polyamines are involved in the phosphorylation of RB, but the inhibition of polyamine biosynthesis by TGF-beta1 did not result in the inhibition of RB phosphorylation and DNA synthesis.
Asunto(s)
Factor de Crecimiento de Hepatocito/farmacología , Hígado/metabolismo , Ornitina Descarboxilasa/metabolismo , Proteína de Retinoblastoma/metabolismo , Animales , Células Cultivadas , Eflornitina/farmacología , Inhibidores Enzimáticos/farmacología , Masculino , Ornitina Descarboxilasa/farmacología , Inhibidores de la Ornitina Descarboxilasa , Fosforilación , Ratas , Ratas Wistar , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Green tea extract and its polyphenolic components have been found to possess anticarcinogenic, antimutagenic, antihypertensive and antihepatotoxic effects, and several mechanisms have been proposed for these effects. In this study, the effects of five tea polyphenols, (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-) epicatechin-3-gallate (ECG), ( -) epicatechin (EC) and (+)-catechin (C), were examined on the viability of Ehrlich ascites tumor cells in vitro and a possible relationship with tyrosine phosphorylation was determined. Proteins extracted from the cells treated with the tea polyphenols were separated by SDS-PAGE, and tyrosine-phosphorylated proteins were detected by immunoblotting with anti-phosphotyrosine antibody and the extent of phosphorylation determined. EGC (100 microM) caused a significant decrease in cell viability to 4.1 +/- 0.2% of the control value, and this correlated with a stimulation of protein tyrosine kinase (PTK) activity. EGCG (100 microM) also caused a slight decrease in cell viability (approximately 70% of the control value) but this and the other polyphenols, which had no effect on cell viability likewise, had no effect on tyrosine phosphorylation. Tyrosine phosphorylations of 42 and 45 kDa proteins were also observed for EGC. Further evaluation of the effect of EGC showed that the activity of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis in cells, decreased significantly as well. A significant correlation has therefore been observed between a cellular event, namely, a reduction in the viability of Ehrlich ascites tumor cells and an association with a tyrosine phosphorylation of 42 and 45 kDa proteins by the polyphenol EGC.
Asunto(s)
Anticarcinógenos/farmacología , Carcinoma de Ehrlich/metabolismo , Proteínas de Neoplasias/metabolismo , Fenoles/farmacología , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Té , Animales , Catequina/análogos & derivados , Catequina/farmacología , Supervivencia Celular/efectos de los fármacos , Flavonoides/farmacología , Cinética , Ratones , Ornitina Descarboxilasa/metabolismo , Fosforilación , Células Tumorales CultivadasRESUMEN
Oral administration of 1 ml of 3.42 M NaCl solution to rats induced spermidine/spermine N1-acetyltransferase (SSAT) activity in gastric mucosa as well as ornithine decarboxylase (ODC) activity. SSAT activity increased and peaked at 5 h and again at 7 h, whereas ODC activity peaked at 6 h. SSAT mRNA also increased after 3.42 M NaCl administration to an extent similar to the increase in SSAT activity at 5 h. Intracellular putrescine level and DNA synthesis were increased by NaCl administration. A polyamine oxidase inhibitor, N,N'-bis(2,3-butadienyl)-1,4-butanediamine (MDL-72527), but not an ODC inhibitor, alpha-difluoromethylornithine (DFMO), inhibited the increases in putrescine level and DNA synthesis at 5 h. The inhibition of DNA synthesis by MDL-72527 was reversed by putrescine administration. In contrast, both MDL-72527 and DFMO inhibited the increase in putrescine level and DNA synthesis at 16.5 h. These findings suggest that putrescine produced from preexistent spermidine by SSAT is responsible for the initial DNA synthesis after mucosal injury induced by NaCl and that both SSAT and ODC are involved in formation of putrescine, which is required for subsequent DNA synthesis.
Asunto(s)
Acetiltransferasas/metabolismo , Mucosa Gástrica/metabolismo , Poliaminas/metabolismo , Acetiltransferasas/biosíntesis , Administración Oral , Animales , Replicación del ADN , Eflornitina/farmacología , Inhibidores Enzimáticos/farmacología , Mucosa Gástrica/enzimología , Masculino , Ornitina Descarboxilasa/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Putrescina/análogos & derivados , Putrescina/metabolismo , Putrescina/farmacología , Ratas , Ratas Wistar , Solución Salina Hipertónica , Poliamino OxidasaRESUMEN
We previously reported that lack of Zn2+ decreased ornithine decarboxylase (ODC) activity without any change in ODC messenger RNA levels and the half-life of ODC activity being about 2-fold more rapid in primary cultured adult rat hepatocytes, suggesting that lack of Zn2+ decreased ODC activity mainly by degrading the enzyme. The present investigations showed that the chelator, diethylenetriamine penta-acetic acid (DTPA), increased the ratio of ODC-antizyme complex to total ODC (about 2-fold) and caused a decrease in antizyme inhibitor, a protein inhibitor of ODC antizyme (about 50%). These results indicate that a restricted Zn2+ availability affects the antizyme-dependent ODC degradation pathway and consequently decreases ODC activity in primary cultured rat hepatocytes.
Asunto(s)
Hígado/enzimología , Ornitina Descarboxilasa/metabolismo , Zinc/administración & dosificación , Animales , Western Blotting , Células Cultivadas , Quelantes/farmacología , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Insulina/farmacología , Ornitina Descarboxilasa/genética , Ácido Pentético/farmacología , ARN Mensajero/metabolismo , Ratas , Zinc/farmacologíaRESUMEN
The cytoprotective effect of green tea extract and its phenolic compounds against 1,4-naphthoquinone-induced hepatotoxicity was evaluated in primary cultured rat hepatocytes. After exposure to 1,4-naphthoquinone, lactate dehydrogenase (LDH) leakage and cell viability were both improved by the presence of the tea extract and tea polyphenols. This cytoprotective effect was related to the structure of tea polyphenols, the galloyl group of (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate being particularly effective. The production of liquid peroxidation by 1,4-naphthoquinone was not inhibited by the tea extract nor by tea polyphenol addition. After 2 h of incubation, the protein thiol concentration was reduced by 1,4-naphthoquinone, but this reduction was prevented by the tea extract and tea polyphenols. The reduction in protein thiol content of the cells closely paralleled the LDH leakage and loss of cell viability. These results suggest that the mechanism of protection by tea polyphenols against 1,4-naphthoquinone-induced toxicity to rat hepatocytes was due to the maintenance of protein thiol levels.
Asunto(s)
Hepatopatías/prevención & control , Naftoquinonas/toxicidad , Fenoles/uso terapéutico , Té , Animales , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas , Interacciones Farmacológicas , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hepatopatías/metabolismo , Masculino , Extractos Vegetales/uso terapéutico , Polímeros/uso terapéutico , Ratas , Ratas Sprague-Dawley , Compuestos de Sulfhidrilo/metabolismoRESUMEN
The modifying effects of dimethylarsinic acid (DMA), the major metabolite of ingested arsenicals in most mammals, on chemical carcinogenesis were investigated using rat in vivo models and reviewed here. In a multi-organ bioassay, rats pretreated with 5 carcinogens were administered DMA at various concentrations in their drinking water. Significantly increased tumor induction due to DMA was observed in the urinary bladder, kidney, liver, and thyroid gland. This was associated with significantly elevated ornithine decarboxylase activity in the kidneys of DMA-treated animals. To estimate the hazard levels of its promoting influence, further examinations were carried out concerned with urinary bladder and liver carcinogenesis. Doses of 25 and 50 ppm, respectively, of DMA were found capable of enhancing lesion development in the two organs. In conclusion, our data indicate that DMA is a carcinogen or promoter in the urinary bladder, liver, kidney and thyroid gland, in line with previous epidemiological findings.