RESUMEN
Mitosis has been traditionally considered a metabolically inactive phase. We have previously shown, however, that extensive alterations in lipids occur as the cells traverse mitosis, including increased de novo fatty acid (FA) and phosphatidylcholine (PtdCho) synthesis and decreased lysophospholipid content. Given the diverse structural and functional properties of these lipids, we sought to study their metabolic fate and their importance for cell cycle completion. Here we show that FA and PtdCho synthesized at the mitotic exit are destined to the nuclear envelope. Importantly, FA and PtdCho synthesis, but not the decrease in lysophospholipid content, are necessary for cell cycle completion beyond G2/M. Moreover, the presence of alternative pathways for PtdCho synthesis renders the cells less sensitive to its inhibition than to the impairment of FA synthesis. FA synthesis, thus, represents a cell cycle-related metabolic vulnerability that could be exploited for combined chemotherapy. We explored the combination of fatty acid synthase (FASN) inhibition with agents that act at different phases of the cell cycle. Our results show that the effect of FASN inhibition may be enhanced under some drug combinations.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ácido Graso Sintasas/antagonistas & inhibidores , Ácidos Grasos/biosíntesis , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Lipogénesis/efectos de los fármacos , Mitosis/efectos de los fármacos , Membrana Nuclear/metabolismo , Fosfatidilcolinas/biosíntesis , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Etopósido/farmacología , Ácido Graso Sintasas/metabolismo , Células HeLa , Humanos , Lipogénesis/fisiología , Lisofosfolípidos/biosíntesis , Lisofosfolípidos/química , Mitosis/fisiología , Membrana Nuclear/efectos de los fármacos , Membrana Nuclear/enzimologíaRESUMEN
To date, many anticancer drugs have been developed by directly or indirectly targeting microtubules, which are involved in cell division. Although this approach has yielded many anticancer drugs, these drugs produce undesirable side effects. An alternative strategy is needed, and targeting mitotic exit may be one alternative approach. Localization of phosphorylated barrier-to-autointegration factor (BAF) to the chromosomal core region is essential for nuclear envelope compartment relocalization. In this study, we isolated brazilin from Caesalpinia sappan Leguminosae and demonstrated that it inhibited BAF phosphorylation in vitro and in vivo. Moreover, we demonstrated direct binding between brazilin and BAF. The inhibition of BAF phosphorylation induced abnormal nuclear envelope reassembly and cell death, indicating that perturbation of nuclear envelope reassembly could be a novel approach to anticancer therapy. We propose that brazilin isolated from C. sappan may be a new anticancer drug candidate that induces cell death by inhibiting vaccinia-related kinase 1-mediated BAF phosphorylation.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Benzopiranos/aislamiento & purificación , Benzopiranos/farmacología , Caesalpinia/química , Proteínas de Unión al ADN/metabolismo , Membrana Nuclear/efectos de los fármacos , Proteínas Nucleares/metabolismo , Animales , Antineoplásicos/metabolismo , Benzopiranos/metabolismo , Muerte Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Membrana Nuclear/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Telofase/efectos de los fármacosRESUMEN
Recent research suggests that crack cocaine use alters systemic biochemical markers, like oxidative damage and inflammation markers, but very few studies have assessed the potential effects of crack cocaine at the cellular level. We assessed genome instability by means of the comet assay and the cytokinesis-block micronucleus technique in crack cocaine users at the time of admission to a rehabilitation clinic and at two times after the beginning of withdrawal. Thirty one active users of crack cocaine and forty control subjects were evaluated. Comparison between controls and crack cocaine users at the first analysis showed significant differences in the rates of DNA damage (p = 0.037). The frequency of micronuclei (MN) (p < 0.001) and nuclear buds (NBUDs) (p < 0.001) was increased, but not the frequency of nucleoplasmic bridges (NPBs) (p = 0.089). DNA damage decreased only after the end of treatment (p < 0.001). Micronuclei frequency did not decrease after treatment, and nuclear buds increased substantially. The results of this study reveal the genotoxic and mutagenic effects of crack cocaine use in human lymphocytes and pave the way for further research on cellular responses and the possible consequences of DNA damage, such as induction of irreversible neurological disease and cancer.
Asunto(s)
Trastornos Relacionados con Cocaína , Cocaína Crack/toxicidad , Inestabilidad Genómica/efectos de los fármacos , Linfocitos/efectos de los fármacos , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Adolescente , Adulto , Brasil , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Ensayo Cometa , Daño del ADN/efectos de los fármacos , Humanos , Linfocitos/citología , Masculino , Pruebas de Micronúcleos , Persona de Mediana Edad , Membrana Nuclear/efectos de los fármacos , Membrana Nuclear/genéticaRESUMEN
We have previously demonstrated antileishmanial activity on Leishmania amazonensis of the natural (1-2), synthetic (7) and derivatives of coumarin (-) mammea A/BB (3-6) isolated from the dichloromethane extract of Calophyllum brasiliense leaves. The aim of the present study was to evaluate morphological and ultrastructural alterations in Leishmania amazonensis induced by these compounds. In promastigote forms, all seven compounds produced significant morphological and ultrastructural alterations, as revealed by scanning and transmission electron microscopy. The compound 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one (3), the most active antileishmanial with LD50 of 0.9 µM), induced cell shrinkage and a rounded appearance of the cells. Parasites incubated in the presence of compound (3) showed ultrastructural changes, such as the appearance of mitochondrial swelling with a reduction in the density of the mitochondrial matrix and the presence of vesicles inside the mitochondrion, indicating damage and significant change in this organelle; abnormal chromatin condensation, alterations in the nuclear envelope, intense atypical cytoplasmic vacuolization, and the appearance of autophagic vacuoles were also observed. In addition, the compound (3) may be acting to depolarize the mitochondrial membrane potential of the cells, leading to death of the parasite.
Asunto(s)
Antiprotozoarios/farmacología , Calophyllum/química , Cumarinas/química , Leishmania mexicana/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Hojas de la Planta/química , Antiprotozoarios/química , Antiprotozoarios/aislamiento & purificación , Cromanos/aislamiento & purificación , Cromanos/farmacología , Cromatina/efectos de los fármacos , Citometría de Flujo , Concentración 50 Inhibidora , Leishmania mexicana/ultraestructura , Potencial de la Membrana Mitocondrial , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Membrana Nuclear/efectos de los fármacos , Pruebas de Sensibilidad Parasitaria , Extractos Vegetales/química , Extractos Vegetales/farmacologíaRESUMEN
The variations of the intracellular localization of the individual protein kinase C (PKC) isoforms are related with their different biological functions. In this study, we have investigated the precise intracellular translocation of endogenous PKCalpha and PKCepsilon in PMA-stimulated normal and tumoral lactotroph cells by using confocal and immunogold electron microscopy, which was correlated with the rate of cell proliferation of both pituitary cell phenotypes. The present results showed that the short phorbol ester incubation stimulated the proliferation of normal and tumoral lactotroph cells, as determined by the measurement of the BrdU-labelling index. The translocation of PKCalpha to plasma and nuclear membranes induced by PMA was more marked than that observed for PKCepsilon in normal and tumoral lactotroph cells. Our results showed that PKCs translocation to the plasma and nuclear membranes varied from isozyme to isozyme emphasizing that PKCalpha could be related with the mitogenic stimulus exerted by phorbol ester. These data support the notion that specific PKC isozymes may exert spatially defined effects by virtue of their directed translocation to distinct intracellular sites.
Asunto(s)
Lactotrofos/enzimología , Lactotrofos/patología , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente , Lactotrofos/efectos de los fármacos , Lactotrofos/ultraestructura , Mitógenos/farmacología , Membrana Nuclear/efectos de los fármacos , Membrana Nuclear/enzimología , Membrana Nuclear/ultraestructura , Neoplasias Hipofisarias/enzimología , Neoplasias Hipofisarias/patología , Neoplasias Hipofisarias/ultraestructura , Proteína Quinasa C-alfa/ultraestructura , Proteína Quinasa C-epsilon/ultraestructura , Transporte de Proteínas/efectos de los fármacos , Ratas , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimologíaRESUMEN
Dietary fat affects metabolic pathways for phospholipid biosynthesis in tissues in a coordinated fashion. This may be important to aspects of development that concern phosphatidylcholine metabolism or regulatory processes that depend on signals from a changing milieu in the microenvironment of the membrane. Dietary fat influences the phosphatidylethanolamine (PE) composition in many membranes of the brain and retina and may by altered by small changes in the content of 20:4(6) and 22:6(3). Membrane PE fatty acids that contain one, four, or six double bonds and the ratio of 22:5(6) to 22:6(3) in PE that contains four to six double bonds are also affected. An increase in the omega 6 fatty acid content of membranes is associated with increased PE methyltransferase activity and decreased phosphocholine transferase activity, thus indicating a mechanism by which change in an exogenous factor (e.g., dietary fat intake) may alter neural phospholipid biosynthesis. Small changes in the composition of dietary fat intake change the composition of brain membranes during development. It is provocative to ponder whether diet could be used to induce formation of membrane structures that are more resistant to specific insults that cause degeneration of brain structural material, to ensure optimal functional compositions, or to reverse degenerative changes that occur in neural membrane structure and function.
Asunto(s)
Encéfalo/metabolismo , Membrana Celular/metabolismo , Grasas Insaturadas en la Dieta/administración & dosificación , Ácidos Grasos Omega-3/administración & dosificación , Ácidos Grasos Insaturados/administración & dosificación , Lípidos de la Membrana/metabolismo , Metiltransferasas/biosíntesis , Membrana Nuclear/metabolismo , Retina/metabolismo , Animales , Animales Recién Nacidos , Ácido Araquidónico/biosíntesis , Encéfalo/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Ácidos Docosahexaenoicos/metabolismo , Ácidos Grasos Omega-6 , Humanos , Lactante , Recién Nacido , Metiltransferasas/efectos de los fármacos , Membrana Nuclear/efectos de los fármacos , Fosfatidiletanolamina N-Metiltransferasa , Retina/efectos de los fármacosRESUMEN
The numbers of nuclear pore complexes and tight junctions were quantified in the seminal vesicle epithelial cells of castrated and castrated-plus-androgen-treated male rats, which received subcutaneous pellets of testosterone propionate (1 mg/kg body weight) for 1 week. Seminal vesicle weights were 0.284 +/- 0.02 g for castrated, 1.006 +/- 0.006 g for androgen-treated, and 0.918 +/- 0.04 g for untreated groups. Tissue samples were processed for light or electron microscopy and for freeze-fracture techniques. Nuclear areas were measured: controls were 279.34 +/- 8 microns 2; these increased significantly (P less than .001) in castrated-plus-androgen-treated rats (324.66 +/- 11 microns 2) and decreased (P less than .001) in castrated animals (173.14 +/- 6.3 microns 2). Nuclear pore density increased (P less than .001) in castrated-plus-androgen-stimulated rats (5.38 +/- 0.24 pores/microns 2) (control: 4.78 +/- 0.14 pores/microns 2), and decreased (P less than .001) in castrated rats (3.16 +/- 0.14 pores/microns 2). A significant (P less than .001) increase in numbers of tight junction strands that extended in the lateral cell membranes was detected in castrated-plus-androgen-treated rats vs. controls or castrated-only animals.
Asunto(s)
Andrógenos/farmacología , Uniones Intercelulares/ultraestructura , Membrana Nuclear/ultraestructura , Vesículas Seminales/ultraestructura , Animales , Epitelio/efectos de los fármacos , Epitelio/ultraestructura , Técnica de Fractura por Congelación , Uniones Intercelulares/efectos de los fármacos , Masculino , Microscopía Electrónica/métodos , Membrana Nuclear/efectos de los fármacos , Tamaño de los Órganos , Ratas , Vesículas Seminales/anatomía & histología , Vesículas Seminales/efectos de los fármacosRESUMEN
Se determinó el efecto que produce la administración de Tioacetamida (TAA) sobre la composición y propiedades de los núcleos y envoltura nuclear (EN) purificados de la corteza renal (CR) de ratas, en relación a preparaciones obtenidas de animales controles a través de un método para la obtención de la EN, cuya pureza relativa fue analizada por microscopía electrónica y métodos enzimáticos. En la fracción nuclear de los animales tratados con TAA, se observaron las siguientes alteraciones: aumento en la concentración de RNA y fosfolípidos (pg/Núcleo) en un 77%, un leve incremento de DNA (pg/Núcleo) en un 17%. Por otro lado la concentración de proteínas (pg/Núcleo) permaneció constante. En la EN se encontró que la concentración de DNA disminuyó en un 68% y la de fosfolípidos en un 37%; en cambio la concentración de RNA aumentó discretamente en un 11%. Se encontraron importantes diferencias al estudiar el patrón polipeptídico obtenido al realizar electroforesis en geles de poliacrilamida de EN preparadas a partir de hígado y CR de ratas controles y tratadas con TAA. La actividad específica de la enzima glucosa-6-fosfatasa en los núcleos y EN de los animales tratados con TAA disminuyó en 54% y 43% respectivamente. En los estudios cinéticos se encontró que por efecto de la droga de la Vmax disminuyó significativamente sin alteración del Km. Se discuten las implicaciones de estos resultados