RESUMEN
Cellular oxidants, called reactive oxygen species (ROS), are constantly produced in animal and human cells. Excessive ROS can induce oxidative damage in cell constituents and promote a number of degenerative diseases and aging. Cellular antioxidants protect against the damaging effects of ROS. However, in moderate concentrations, ROS are necessary for a number of protective reactions. Thus, ROS are essential mediators of antimicrobial phagocytosis, detoxification reactions carried out by the cytochrome P-450 complex, and apoptosis which eliminates cancerous and other life-threatening cells. Excessive antioxidants could dangerously interfere with these protective functions, while temporary depletion of antioxidants can enhance anti-cancer effects of apoptosis. Experimental data are presented supporting these notions. The human population is heterogeneous regarding ROS levels. Intake of exogenous antioxidants (vitamins E, C, beta-carotene and others) could protect against cancer and other degenerative diseases in people with innate or acquired high levels of ROS. However, abundant antioxidants might suppress these protective functions, particularly in people with a low innate baseline level of ROS. Screening human populations for ROS levels could help identify groups with a high level of ROS that are at a risk of developing cancer and other degenerative diseases. It also could identify groups with a low level of ROS that are at a risk of down-regulating ROS-dependent anti-cancer and other protective reactions. Screening populations could provide a scientifically grounded application of antioxidant supplements, which could significantly contribute to the nation's health.
Asunto(s)
Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Antioxidantes/efectos adversos , Antioxidantes/uso terapéutico , Neoplasias/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , División Celular , Humanos , Tamizaje Masivo , Estrés Oxidativo , Resultado del TratamientoRESUMEN
The mechanism of induction of apoptosis by the novel anti-cancer drug 1-O-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) was investigated in p53-defective SV40 immortalized rat hepatocytes (CWSV1). Exposure to 12 microM ET-18-OCH3 for 36 h induced apoptosis as determined using classical morphological features and agarose gel electrophoresis of genomic DNA. Increased levels of reactive oxygen species (ROS) were detected spectrophotometrically using a nitroblue tetrazolium (NBT) assay in cells treated with ET-18-OCH3. Both the increased generation of ROS and the induction of apoptosis were inhibited when cells were treated concurrently with ET-18-OCH3 in the presence of the antioxidant alpha-tocopherol. Similar results were achieved when cells were switched acutely to choline-deficient (CD) medium in the presence of the antioxidant. The possible role of mitochondria in the generation of ROS was investigated. Both ET-18-OCH3 and CD decreased the phosphatidylcholine (PC) content of mitochondrial and associated membranes, which correlated with depolarization of the mitochondrial membrane as analyzed using 5,5',6,6'-tetramethylbenzimidazolcarbocyanine iodide (JC-1), a sensitive probe of mitochondrial membrane potential. Rotenone, an inhibitor of the mitochondrial electron transport chain, significantly reduced the intracellular level of ROS and prevented mitochondrial membrane depolarization, correlating with a reduction of apoptosis in response to either ET-18-OCH3 or CD. Taken together, these results suggest that the form of p53-independent apoptosis induced by ET-18-OCH3 is mediated by alterations in mitochondrial membrane PC, a loss of mitochondrial membrane potential, and the release of ROS, resulting in completion of apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Hepatocitos/fisiología , Mitocondrias Hepáticas/fisiología , Éteres Fosfolípidos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Animales , Antígenos Transformadores de Poliomavirus/farmacología , Antioxidantes/farmacología , Línea Celular Transformada , Electroforesis en Gel de Agar , Hepatocitos/ultraestructura , Membranas Intracelulares/química , Membranas Intracelulares/fisiología , Masculino , Potenciales de la Membrana , Mitocondrias Hepáticas/química , Mitocondrias Hepáticas/ultraestructura , Nitroazul de Tetrazolio , Fosfatidilcolinas/análisis , Ratas , Ratas Endogámicas F344 , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Vitamina E/farmacologíaRESUMEN
During a study involving treatment of MCF-7 culture of mammary tumor cells with cisplatin 5.10, 15.20 and 25 microM, an inverse correlation was found between the first two doses and the number of dead cells; later on, this fraction diminished gradually to basal level. Conversely, the fraction of necrotized cells increased in proportion to dosage, overall toxicity of cisplatin (the sum total of dead cells) remaining unchanged when in excess of 10 microM.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Cisplatino/farmacología , Apoptosis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Necrosis , Células Tumorales CultivadasRESUMEN
Apoptosis, or regulated cell suicide, eliminates unwanted and damaged cells, including precancerous and cancerous cells. Since reactive oxygen species (ROS) act as essential apoptotic mediators, we reasoned that increasing the ROS level might enhance apoptosis and thereby slow down tumor growth. Here, using a defined transgenic brain tumor model with known tumor apoptosis rates, we test the impact of antioxidant-depleted diet, capable of increasing ROS levels, or antioxidant-enriched diets on tumor growth. Dramatically increased apoptosis occurs within tumors, but not in normal tissues of antioxidant-depleted mice. The presence of detectable increased oxidant stress within tumors indicates that the likely mechanism of enhanced tumor apoptosis is via ROS and DNA oxidative impairment. Importantly, due to the ROS-enhanced apoptosis, tumor growth is inhibited in mice fed an antioxidant-depleted diet. In clear contrast, an antioxidant-rich diet had no impact on tumor growth.
Asunto(s)
Antioxidantes/administración & dosificación , Apoptosis , Neoplasias Encefálicas/patología , Dieta , Animales , Neoplasias Encefálicas/metabolismo , División Celular , Ratones , Ratones Transgénicos , Estrés Oxidativo , Especies Reactivas de OxígenoRESUMEN
Choline deficiency (CD) was previously shown to trigger apoptosis in rat hepatocytes in culture and in vivo. In the present study we investigated the effects of short-term withdrawal of choline from the diet on the expression of putative preneoplastic foci in OXYS rats, an inbred strain with an inherited overproduction of free radicals. Animals were fed a defined, choline-sufficient (CS, control) or choline-deficient (CD) diet for 6 weeks. Eosinophilic, glutathione S-transferase (pi class) (+) preneoplastic foci were found in histologic sections of control OXYS rat liver. CD caused a 60% decrease in the number of eosinophilic foci per liver section (27.0+/-6.1 vs. 10.6+/-4.6 foci/section) compared to CS controls. Apoptotic bodies were detected in 0.18+/-0.03% of hepatocytes in CD livers compared to 0.05+/-0.009% of hepatocytes in controls. Cells which exhibited an apoptotic morphology in hematoxylin and eosin-stained sections were TUNEL-positive, confirming the induction of apoptosis. Also in CD animals compared to controls, there was an increased expression of p27Kip1 protein, and a reduction in PCNA nuclear labeling and the number of mitotic figures, consistent with an inhibition of cell proliferation in the livers of CD animals. This study shows that the liver of OXYS rats with an inherited overgeneration of free radicals retains sensitivity to CD, and that this p53-independent trigger of apoptosis can decrease the number of eosinophilic foci in the livers of these animals.
Asunto(s)
Apoptosis , Deficiencia de Colina/patología , Hígado/patología , Animales , División Celular , Eosinófilos/patología , Radicales Libres/metabolismo , Hígado/metabolismo , Neoplasias Hepáticas Experimentales/etiología , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/prevención & control , Masculino , Lesiones Precancerosas/etiología , Lesiones Precancerosas/patología , Lesiones Precancerosas/prevención & control , Ratas , Ratas EndogámicasRESUMEN
On the basis of experimental studies of the initial stages of glycine oligomerization in aqueous suspension of zeolite and kaolinite catalysts, a model is suggested for the prebiotic synthesis of oligopeptides from alpha-amino acids. The formation of linear dipeptides by hydrolysis of one amide bond in the cyclic piperazinedione intermediate (formed from glycine spontaneously) is found to be the critical stage of the reaction. This stage is base catalyzed and its rate increases when pH of the medium goes up. The linear glycyl-glycine yield rises under effect of hydroxyl anions generated from different sources including insoluble silicates and soluble sodium bicarbonate. During prebiotic evolution silicates capable of cation-exchange can serve as local sources of the hydroxyl anions which dramatically accelerate formation of linear dipeptides from cyclic ones. Oligopeptides of higher molecular weight are then easily formed from the linear dipeptides at neutral pH, even in the absence of catalysts or sources of energy (e.g. such as light). The described catalytic synthesis could occur in the proximity of submarine hydrothermal vents.
Asunto(s)
Evolución Molecular , Glicina/química , Glicilglicina/química , Oligopéptidos/síntesis química , Piperazinas/síntesis química , Silicatos/química , Silicatos de Aluminio/química , Catálisis , Arcilla , Evolución Química , Concentración de Iones de Hidrógeno , Origen de la Vida , Zeolitas/síntesis química , Zeolitas/químicaRESUMEN
The mechanisms which drive initiated cells to progress to form carcinomas are poorly understood. CWSV-1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, respond by inducing p53-independent apoptosis when acutely switched to medium containing low choline (16% apoptotic at 48 h in 5 microM choline) as compared with controls (1% apoptotic at 48 h in 70 microM choline). The rate of apoptosis was inversely correlated with cellular phosphatidylcholine content. Choline deficiency (CD)-induced apoptosis is probably mediated by TGFbeta1 and reactive oxygen species, since immunoneutralization of TGFbeta1 in the medium or treatment with N-acetylcysteine (an antioxidant) or addition of neocuproine (a transition metal chelator) prevented CD-induced apoptosis. CWSV-1 hepatocytes could be gradually adapted to survive in 5 microM choline. CD-adapted cells had increased membrane phosphatidylcholine concentrations (compared with acute CD cells). Adapted cells acquired relative resistance to CD-induced apoptosis (7% of adapted cells compared with 19% of non-adapted cells were apoptotic at 48 h in 5 microM choline). They also became relatively resistant to another p53-independent form of apoptosis (TGFbeta1-induced). CD-adapted hepatocytes developed increased capability for anchorage-independent growth and formed tumors when transplanted into nude mice; passage-matched control hepatocytes did not possess these properties. Cell transformation was dependent on exposure to the selective pressure of CD apoptosis, as we observed that when CD apoptosis was inhibited with an antioxidant during adaptation, cells did not become anchorage independent. Acquisition by p53-deficient cells of resistance to p53-independent inducers of apoptosis (CD, TGFbeta1 and reactive oxygen species) may leave cells without another important apoptotic defensive barrier and may be responsible for the progression of initiated cells to frank carcinomas.
Asunto(s)
Apoptosis/genética , Transformación Celular Neoplásica/genética , Deficiencia de Colina/patología , Genes p53 , Hígado/patología , Animales , Deficiencia de Colina/genética , Masculino , Ratones , Ratones Desnudos , Ratas , Ratas Endogámicas F344RESUMEN
It is known that long-term withdrawal of choline from the diet induces hepatocellular carcinomas in animal models in the absence of known carcinogens. We hypothesize that a choline deficient diet (CD) alters the balance of cell growth and cell death in hepatocytes and thus promotes the survival of clones of cells capable of malignant transformation. When grown in CD medium (5 microM or 0 microM choline) CWSV-1 rat hepatocytes immortalized with SV40 large T-antigen underwent p53-independent apoptosis (terminal dUTP end-labeling of fragmented DNA; laddering of DNA in agarose gel). CWSV-1 cells which were adapted to survive in 5 microM choline acquired resistance to CD-induced apoptosis and were able to form hepatocellular carcinomas in nude mice. These adapted CWSV-1 cells express higher amounts of both the 32 kDa membrane-bound and 6 kDa mature form of TGF alpha compared to cells made acutely CD. Control (70 microM choline) and adapted cells, but not acutely deficient hepatocytes, could be induced to undergo apoptosis by neutralization of secreted TGF alpha. Protein tyrosine phosphorylation is known to protect against apoptosis. We found decreased EGF receptor tyrosine phosphorylation in acutely choline deficient CWSV-1 cells. TGF beta 1 is an important growth-regulator in the liver. CWSV-1 cells express TGF beta 1 receptors and this peptide induced cell detachment and death in control and acutely deficient cells. Hepatocytes adapted to survive in low choline were also resistant to TGF beta 1, although TGF beta 1 receptors and protein could be detected in the cytoplasm of these cells. The non-essential nutrient choline is important in maintaining plasma membrane structure and function, and in intracellular signaling. Our results indicate that acute withdrawal of choline induces p53-independent programmed cell death in hepatocytes, whereas cells adapted to survive in low choline are resistant to this form of apoptosis, as well as to cell death induced by TGF beta 1. Our results also suggest that CD may induce alterations (mutations?) in growth factor signaling pathways which may enhance cell survival and malignant transformation.
Asunto(s)
Apoptosis , Deficiencia de Colina/metabolismo , Dieta , Neoplasias/etiología , Animales , Colina/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Humanos , Transducción de Señal , Factor de Crecimiento Transformador alfa/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
Immortalized CWSV-1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, had increased numbers of cells with strand breaks in genomic DNA (terminal dUTP end labeling) when grown in 0 Micron choline (67-73% of cells) than when grown in 70 Micron choline (2-3% of cells). Internucleosomal fragmentation of DNA (DNA ladders) was detected in cells grown with 5 Micron and 0 Micron choline for 72h. Cells treated with 0 or 5 Micron choline for 72h detached from the substrate in high numbers (58% of choline deficient cells vs. 1.4% of choline sufficient cells detached) exhibited a high incidence of apoptosis (apoptotic bodies were seen in 55-75% of cells; 67-73% had DNA strand breaks), and an absence of mitosis and proliferating cell nuclear antigen (PCNA) expression. Cells undergoing DNA fragmentation had functioning mitochondria. At 24h, cells grown in 0 or 5 Micron choline synthesize DNA more rapidly than those grown in 70 Micron choline. By 72h, the cells grown in 0 or 5 Micron choline were forming DNA much more slowly than control cells (assessed by thymidine incorporation, PCNA expression, and mitotic index). Western blot analysis showed that p53 in the nucleus of cells was detected in direct association with SV40 T-antigen, and was therefore likely to be inactive. We conclude that choline deficiency kills CWSV-1 hepatocytes in culture by inducing apoptosis via what may be a p53-independent process, and that this process begins in viable cells before they detach from the culture dish.
Asunto(s)
Apoptosis , Deficiencia de Colina/patología , Hígado/patología , Virus 40 de los Simios/genética , Animales , División Celular , Supervivencia Celular , Transformación Celular Viral , Células Cultivadas , ADN/metabolismo , Daño del ADN , Ratas , Proteína p53 Supresora de Tumor/análisisAsunto(s)
Peroxidación de Lípido , Errores Innatos del Metabolismo/metabolismo , Especies Reactivas de Oxígeno , Vitamina E/metabolismo , Tejido Adiposo/metabolismo , Glándulas Suprarrenales/metabolismo , Animales , Epidídimo/metabolismo , Masculino , Malondialdehído/sangre , Mitocondrias Hepáticas/metabolismo , Miocardio/metabolismo , Ratas , Ratas Wistar , Vitamina E/sangreAsunto(s)
Antimaláricos/farmacología , Cloroquina/farmacología , ADN Protozoario/efectos de los fármacos , Plasmodium berghei/efectos de los fármacos , Plasmodium berghei/genética , ARN Protozoario/efectos de los fármacos , Animales , Sistema Enzimático del Citocromo P-450/genética , ADN Complementario , Resistencia a Medicamentos/genética , Regulación Enzimológica de la Expresión GénicaRESUMEN
The rates of oxygen consumption in liver mitochondria of strain S rats (2-3- and 10-12-months-old) with inherited free radical hyperproduction during oxidation of three types of substrates (glutamate+maleate, succinate+rotenone and ascorbate+TMPD) as well as cytochrome a level in the respiratory chain are lowered in comparison with Wistar controls. In liver mitochondria of 10-12-months-old rats these changes are more pronounced and manifest themselves as decreased ADP/O, phosphorylation rates and F0F1-ATPase activity. The phosphorylation potential, ATP content and adenine nucleotide pool in rat liver are also reduced. The experimental results give a better insight into the pathogenetic mechanisms of morbid states (e.g., degenerative diseases) in human beings and shed additional light on the origin of short lifespan and premature in strain S rats.
Asunto(s)
Citocromos/metabolismo , Radicales Libres , Mitocondrias Hepáticas/enzimología , ATPasas de Translocación de Protón/metabolismo , Animales , Transporte de Electrón , Cinética , Masculino , Mitocondrias Hepáticas/metabolismo , Fosforilación Oxidativa , Oxígeno/metabolismo , Ratas , Ratas WistarAsunto(s)
Cardiomiopatías/metabolismo , Envejecimiento/metabolismo , Animales , Transporte Biológico , Cardiomiopatías/enzimología , Cardiomiopatías/genética , Membrana Celular/metabolismo , Desoxiglucosa/metabolismo , Glicósido Hidrolasas/metabolismo , Hexosas/metabolismo , Hidrolasas/metabolismo , Radical Hidroxilo/metabolismo , Peroxidación de Lípido , Masculino , Mitocondrias Cardíacas/metabolismo , Ratas , TritioAsunto(s)
Membranas Intracelulares/metabolismo , Errores Innatos del Metabolismo/metabolismo , Mitocondrias Hepáticas/metabolismo , Envejecimiento/metabolismo , Animales , Transporte de Electrón , Radicales Libres , Masculino , Fosforilación Oxidativa , Estrés Oxidativo , Ratas , Ratas Wistar , Especificidad de la EspecieAsunto(s)
Carcinoma Krebs 2/patología , Ciclofosfamida/farmacología , Ultrasonido , Animales , División Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Resistencia a Medicamentos , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Peroxidación de Lípido , Ratones , Marcadores de Spin , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The functional characteristics of liver mitochondria and physical-chemical properties of mitochondrial membranes were studied in S rats with congenitally enhanced capacity to free radical generation in comparison to those in Wistar rats. It was shown previously that intense lipid peroxidation, numerous DNA rearrangements, protein oxidation, morbid states resembling human degenerative diseases and short life-span are characteristic of S rats. In present study we have demonstrated that in S rats at the age of 2-3 months the respiratory rate of the mitochondria in active metabolic state is lower than in Wistar rats, and so are the values for the respiratory control ratio, oxidative phosphorylation and membrane potential. By 10-12 months of age the decrease of the respiration rate and oxidative phosphorylation in S rat mitochondria become even more dramatic. These changes are associated with a decrease in the extent of dip of proteins into the membrane lipid layer or with the increase in the amount of protein aggregates. The results add to the understanding of the nature of morbid conditions developed under the effect of intensive free radical generation and to the comprehension of their role in aging.