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This study explores the impact of serious illnesses, such as cancer, on patients' time preferences in medical decision-making. Specifically, we assess how patients value extending their lifespan by one year under varying survival prognoses through three experimental studies. The findings reveal that patients exhibit a higher Subjective Discount Rates (SDR) in their medical decisions after a serious illness diagnosis. Notably, this difference in individual health also affects the time preferences of their family members. Additionally, the subjective contextual setting of the illness can also increase an individual's SDR levels. The research highlights a tendency for patients and families facing a potential short life expectancy to focus more on immediate concerns, leading to potentially shortsighted and irrational medical choices. This behavior often results in regret during the end-of-life stage. These insights are vital for healthcare professionals in optimizing treatment plans and for policymakers in understanding patient behaviors more comprehensively. The study emphasizes the need for considering psychological and behavioral changes in patients grappling with severe health challenges.
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BRISC (BRCC3 isopeptidase complex) is a deubiquitinating enzyme that has been linked with inflammatory processes, but its role in liver diseases and the underlying mechanism are unknown. Here, we investigated the pathophysiological role of BRISC in acute liver failure using a mice model induced by D-galactosamine (D-GalN) plus lipopolysaccharide (LPS). We found that the expression of BRISC components was dramatically increased in kupffer cells (KCs) upon LPS treatment in vitro or by the injection of LPS in D-GalN-sensitized mice. D-GalN plus LPS-induced liver damage and mortality in global BRISC-null mice were markedly attenuated, which was accompanied by impaired hepatocyte death and hepatic inflammation response. Constantly, treatment with thiolutin, a potent BRISC inhibitor, remarkably alleviated D-GalN/LPS-induced liver injury in mice. By using bone marrow-reconstituted chimeric mice and cell-specific BRISC-deficient mice, we demonstrated that KCs are the key effector cells responsible for protection against D-GalN/LPS-induced liver injury in BRISC-deficient mice. Mechanistically, we found that hepatic and circulating levels of TNF-α, IL-6, MCP-1, and IL-1ß, as well as TNF-α- and MCP-1-producing KCs, in BRISC-deleted mice were dramatically decreased as early as 1 h after D-GalN/LPS challenge, which occurred prior to the elevation of the liver injury markers. Moreover, LPS-induced proinflammatory cytokines production in KCs was significantly diminished by BRISC deficiency in vitro, which was accompanied by potently attenuated NF-κB activation. Restoration of NF-κB activation by two small molecular activators of NF-κB p65 effectively reversed the suppression of cytokines production in ABRO1-deficient KCs by LPS. In conclusion, BRISC is required for optimal activation of NF-κB-mediated proinflammatory cytokines production in LPS-treated KCs and contributes to acute liver injury. This study opens the possibility to develop new strategies for the inhibition of KCs-driven inflammation in liver diseases.
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Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Enfermedad Hepática Inducida por Sustancias y Drogas , Animales , Ratones , FN-kappa B/metabolismo , Lipopolisacáridos/farmacología , Macrófagos del Hígado/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Hígado/metabolismo , Inflamación/metabolismo , Galactosamina , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismoRESUMEN
We present a simple and robust LC-MS/MS assay for the simultaneous quantitation of an antibody cocktail of trastuzumab and pertuzumab in monkey serum. The LC-MS/MS method saved costs, decreased the analysis time, and reduced quantitative times relative to the traditional ligand-binding assays. The serum samples were digested with trypsin at 50°C for 60 min after methanol precipitation, ammonium bicarbonate denaturation, dithiothreitol reduction, and iodoacetamide alkylation. The tryptic peptides were chromatographically separated using a C18 column (2.1 × 50 mm, 2.6 µm) with mobile phases of 0.1% formic acid in water and acetonitrile. The other monoclonal antibody, infliximab, was used as internal standards to minimize the variability during sample processing and detection. A unique peptide for each monoclonal antibody was simultaneously quantified using LC-MS/MS in the multiple reaction monitoring mode. Calibration curves were linear from 2.0 to 400 µg/mL. The intra- and inter-assay precision (%CV) was within 8.9 and 7.4% (except 10.4 and 15.1% for lower limit of quantitation), respectively, and the accuracy (%Dev) was within ±13.1%. The other validation parameters were evaluated, and all results met the acceptance criteria of the international guiding principles. Finally, the method was successfully applied to a pharmacokinetics study after a single-dose intravenous drip administration to cynomolgus monkeys.
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Anticuerpos Monoclonales Humanizados/sangre , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Trastuzumab/sangre , Animales , Anticuerpos Monoclonales Humanizados/farmacocinética , Femenino , Modelos Lineales , Macaca fascicularis , Masculino , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Trastuzumab/farmacocinéticaRESUMEN
Danshensu [3-(3, 4-dihydroxyphenyl) lactic acid, DSS], one of the significant cardioprotective components, is extracted from the root of Salvia miltiorrhiza. In the present study, an ester prodrug of Danshensu (DSS), palmitoyl Danshensu (PDSS), was synthesized with the aim to improve its oral bioavailability and prolong its half-life. The in vitro experiments were carried out to evaluate the physicochemical properties and stability of PDSS. Although the solubility of PDSS in water was only 0.055 mg·mL-1, its solubility in FaSSIF and FeSSIF reached 4.68 and 9.08 mg·mL-1, respectively. Octanol-water partition coefficient (log P) was increased from -2.48 of DSS to 1.90 of PDSS. PDSS was relatively stable in the aqueous solution in pH range from 5.6 to 7.4. Furthermore, the pharmacokinetics in rats was evaluated after oral administration of PDSS and DSS. AUC and t1/2 of PDSS were enhanced up to 9.8-fold and 2.2-fold, respectively, compared to that of DSS. Cmax was 1.67 ± 0.11 µg·mL-1 for PDSS and 0.81 ± 0.06 µg·mL-1 for DSS. Thus, these results demonstrated that PDSS had much higher oral bioavailability and longer circulation time than its parent drug.
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Composición de Medicamentos/métodos , Lactatos/química , Profármacos/química , Salvia miltiorrhiza/química , Animales , Disponibilidad Biológica , Evaluación Preclínica de Medicamentos , Concentración de Iones de Hidrógeno , Lactatos/farmacocinética , Masculino , Profármacos/farmacocinética , Ratas , Ratas Sprague-Dawley , SolubilidadRESUMEN
α-Chymotrypsin (α-CT) and trypsin are important components of the enzymatic barrier. They could degrade the therapeutic proteins and peptides, inhibit their activity consequently, and thereby reduce their oral bioavailability. Acidic agents, as one type of indirect protease inhibitors, have shown proof of concept in clinical trials. We report here the inactivated proteases due to acid influence can be reactivated immediately by environmental pH recovery regardless of how long the inactivation last. To keep the inactivation time of proteases for 4-5 h, we designed and prepared a sustained-release tablet containing citric acid (CA) which can effectively reduce the pH below 5.0 and maintain it for 5 h in the dissolution-reaction medium. The activity of α-CT and trypsin was quantified by analyzing the residual amount of their respective substrates BTEE and TAME. More than 80% of the substrates were survived in 5.0 h of incubation, whereas the common tablet inhibited the proteases activity for only two hours in the same experimental medium. It indicates that the sustained-release tablet loaded with CA can efficiently inhibit the α-CT and trypsin activity longer than the common tablet. The results will be beneficial for designing and formulating the peroral administration of peptide and protein drugs.
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Ácidos/química , Quimotripsina/química , Mucosa Intestinal/metabolismo , Péptidos/metabolismo , Inhibidores de Proteasas/metabolismo , Proteínas/metabolismo , Tripsina/química , Concentración de Iones de Hidrógeno , Intestinos/química , Cinética , Péptidos/química , Inhibidores de Proteasas/química , Proteínas/químicaRESUMEN
Toll-like receptor-5 (TLR5) signaling regulates the immune privileged status of the liver and is involved in hepatic immune disorders. However, the role of TLR5 has not yet been investigated in experimental models of concanavalin A (Con A)-mediated liver injury. Here, we show that TLR5 is highly up-regulated in the hepatic mononuclear cells of mice during Con A-induced hepatitis. Increased mortality and liver histopathology of TLR5-deficient mice correlated with excessive production of proinflammatory cytokines, suggesting that TLR5 knockout mice were more susceptible to Con A-induced hepatitis. We also report that administration of CBLB502, an exogenous TLR5 agonist, substantially alleviated Con A-mediated hepatitis in wild-type mice as shown by increased survival rates, reduced aminotransferase and proinflammatory cytokine production, impaired lymphocyte infiltration, and ameliorated hepatocyte necrosis and/or apoptosis. Mechanistic studies revealed that CBLB502 acts as a negative regulator in limiting T-cell/natural killer T-cell activity and cytokine production in the Con A-hepatitis model. Bone marrow transplantation experiments showed that TLR5 in bone marrow-derived cells contributed to the hepatoprotective efficacy of CBLB502 against Con A-induced liver injury. Moreover, interleukin-6 elevation induced by CBLB502 is an important protective factor against Con A-induced liver injury. In addition, we demonstrate that CBLB502 suppresses α-galactosylceramide-induced natural killer T cell-dependent inflammatory liver injury. CONCLUSION: The TLR5 signaling pathway plays an important role in T cell-mediated hepatic injury and may be exploited for therapeutic treatment of inflammatory liver diseases. (Hepatology 2017;65:2059-2073).
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Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Concanavalina A/toxicidad , Células T Asesinas Naturales/inmunología , Péptidos/farmacología , Receptor Toll-Like 5/metabolismo , Animales , Biopsia con Aguja , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/mortalidad , Concanavalina A/farmacología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Inmunohistoquímica , Mediadores de Inflamación/sangre , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Distribución Aleatoria , Valores de Referencia , Transducción de Señal , Tasa de Supervivencia , Receptor Toll-Like 5/efectos de los fármacosRESUMEN
Catalase is a promising therapeutic enzyme; however, it carries risks of inactivation and rapid degradation when it is used in practical bioprocess, such as delivery in vivo. To overcome the issue, we made catalase-only nanoparticles using shear stress alone at a moderate shear rate of 217s(-1) in a coaxial cylinder flow cell. Properties of nanoparticles, including particle size, polydispersity index and zeta potential, were characterized. The conformational changes of pre- and post-sheared catalase were determined using spectroscopy techniques. The results indicated that the conformational changes of catalase and reduction in α-helical content caused by shear alone were less significant than that by desolvation method. Catalase-only nanoparticles prepared by single shear retained over 90% of its initial activity when compared with the native catalase. Catalase nanoparticles lost only 20% of the activity when stored in phosphate buffer solution for 72h at 4°C, whereas native catalase lost 53% under the same condition. Especially, the activity of nanogranulated catalase was decreased only slightly in the simulated intestinal fluid containing α-chymotrypsin during 4h incubation at 37°C, implying that the catalase nanoparticle was more resistant to the degradation of proteases than native catalase molecules. Overall, catalase-only nanoparticles offered a great potential to stabilize enzymes for various pharmaceutical applications.
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Catalasa/metabolismo , Nanopartículas/química , Resistencia al Corte , Animales , Bovinos , Dicroismo Circular , Estabilidad de Enzimas , Nanopartículas/ultraestructura , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier , Factores de TiempoRESUMEN
BACKGROUND & AIMS: Hepassocin (HPS) is a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage. In this paper, zebrafish were used to investigate the role of HPS in liver development. METHODS AND RESULTS: During zebrafish development, HPS expression is enriched in liver throughout hepatogenesis. Knockdown of HPS using its specific morpholino leads to a smaller liver phenotype. Further results showed that the HPS knockdown has no effect on the expression of the early endoderm marker gata6 and early hepatic marker hhex. In addition, results showed that the smaller-liver phenotype in HPS morphants was caused by suppression of cell proliferation, not induction of cell apoptosis. CONCLUSIONS: Current findings indicated that HPS is essential to the later stages of development in vertebrate liver organogenesis.
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Péptidos y Proteínas de Señalización Intercelular/metabolismo , Hígado/embriología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Proliferación Celular , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hepatocitos/citología , Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Hígado/metabolismo , Morfolinos/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Hepassocin (HPS) is a secreted protein with mitogenic activity on primary hepatocytes and protects hepatocytes from chemically-induced injury. Our previous studies showed that HPS stimulates proliferation of hepatocytes in an ERK pathway-dependent manner. However, the molecular mechanism of HPS-induced activation of the ERK pathway remains unclear. In this study, we found that HPS induced the phosphorylation of the epidermal growth factor receptor (EGFR) in the human L02 hepatocyte cell line, and this event was concomitant with the activation of the non-receptor tyrosine kinase Src. Specific inhibition of EGFR kinase activity by gefitinib or down-regulation of EGFR by specific EGFR siRNAs prevented HPS-induced activation of the ERK pathway and proliferation of L02 cells. Furthermore, inhibition of Src activity significantly blocked HPS-induced activation of the EGFR, which was suggestive of a ligand-independent transactivation mechanism of EGFR itself as well as ERK phosphorylation and proliferation of L02 cells. These results indicate that EGFR plays an important role in the mitogenic signaling induced by HPS in L02 cell lines and may further stimulate research on the role of HPS in hepatocytes within biological processes in human health and disease.
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Proliferación Celular/efectos de los fármacos , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas de Neoplasias/farmacología , Familia-src Quinasas/metabolismo , Línea Celular , Regulación hacia Abajo , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Fibrinógeno , Gefitinib , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Quinazolinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Idebenone (IDB) is a synthetic antioxidant and analog of coenzyme Q10. The percutaneous permeation of IDB was investigated in guinea pig skin after application of different formulations. The enhancing effects of various formulations [nanostructured lipid carriers (NLCs), nanoemulsion (NE), or oil solution] on the permeation of IDB were evaluated using ex vivo guinea pig skins. Furthermore, stability of different formulations and in which chemical stability of IDB was determined during storage. Permeation experiments revealed that formulations varied in their ability to enhance the skin permeation of IDB. For NLC formulation, the cumulative amount of IDB in the epidermis, dermis, and acceptor medium of diffusion cells was approximately threefold more than NE or oil solution at the end of 24-h experiment. No significant difference between NE and oil solution was observed in the enhancement of penetration efficacy of IDB. Different formulations resulted in stability with different properties. NLC formulation revealed preferentially more stable than NE. The residual percentage of IDB loaded in NLCs, NE, and oil solution was 90.1%, 65.4%, and 51.3%, respectively, when stored at 40°C under 75% RH and 3,000 lx light conditions for 180 days. The results obtained here demonstrated that the abilities of NLCs to improve the chemical stability of IDB and enhance the skin permeation are much better than NE and oil solution. These suggest that NLCs containing IDB have significant potential use for skin care as an alternative topical formulation.
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Antioxidantes/química , Portadores de Fármacos/química , Lípidos/química , Nanoestructuras/química , Absorción Cutánea , Ubiquinona/análogos & derivados , Animales , Antioxidantes/administración & dosificación , Antioxidantes/metabolismo , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/metabolismo , Estabilidad de Medicamentos , Femenino , Cobayas , Lípidos/administración & dosificación , Lípidos/farmacocinética , Nanoestructuras/administración & dosificación , Absorción Cutánea/efectos de los fármacos , Absorción Cutánea/fisiología , Ubiquinona/administración & dosificación , Ubiquinona/química , Ubiquinona/metabolismoRESUMEN
Solid lipid nanoparticles (SLNs) have attracted increasing attention as colloidal drug carriers due to theirs advantages including low toxicity, drug targeting and modified release. However, undesired particle aggregation in aqueous dispersions would limit the applicability of SLNs for drug delivery. The purpose of the present article is to investigate the aggregation behavior of the SLNs and quantitatively evaluate how the concentration of NaCl and F68 affect the stability of the SLNs. The early stage aggregation kinetics of the SLNs was investigated over a wide range of NaCl concentrations by employing dynamic light scattering (DLS). In the presence of the NaCl, aggregation kinetics of the SLNs exhibited reaction-limited (slow) and diffusion-limited (fast) regimes. These results indicated that the aggregation behavior of these new nanoparticles can be well explained by the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The critical coagulation concentration (CCC) of SLNs containing 0.0%, 0.1%, 0.5%, 2.0%, and 4.0% of Poloxamer 188 (F68) was 416, 328, 519, 607, and 602 mM, respectively, suggesting that the F68 influences the aggregation behavior of the SLNs. F68 made the SLNs more sensitive to the electrolyte when its concentration is low (0.1%), the bush of the polymer F68 has a bridging effect that accelerated the aggregation process of the SLNs. However, at the high concentration, F68 can provide the steric repulsion to the nanoparticles, which effectively stabilized the SLNs dispersions.
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Electrólitos/química , Excipientes/química , Lípidos/química , Nanopartículas/química , Poloxámero/química , Cloruro de Sodio/química , Tensoactivos/química , Adhesividad , Coloides , Difusión , Portadores de Fármacos/química , Ácidos Grasos no Esterificados/química , Cinética , Microscopía Electrónica de Rastreo , Nanopartículas/ultraestructura , Nefelometría y Turbidimetría , Concentración Osmolar , Ácido Palmítico/química , Electricidad Estática , Ácidos Esteáricos/química , Propiedades de SuperficieRESUMEN
There is little information about the inhibitory effects of hyaluronic acid with paclitaxel on tumor metastasis and ascites formation. The aim of present study is to determine whether the combined administration of hyaluronic acid (HA) with paclitaxel can produce additional or synergistic therapeutic effects in the control of Lewis lung carcinoma (LLC) migration and ascites formation of U14 cervical tumor. The anti-metastasis effect of hyaluronic acid alone, paclitaxel alone and paclitaxel-hyaluronic acid combination were examined in a mouse model bearing LLC cells. i.v. Paclitaxel-hyaluronic acid, in the ratio of 1:3 (w/w), significantly reduced the migration of LLC cells in a synergistic fashion. In the experiment with mice bearing U14 tumor cells, i.p. paclitaxel-hyaluronic acid markedly improved the life span of mice and significantly decreased ascites formation when compared to paclitaxel alone or hyalunonan alone. We also analyzed the serum proteome of mice with lung neoplasm before and after treatment with paclitaxel-hyaluronic acid. Proteomic analysis on LLC mice was studied using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. The results showed that i.v. administration of hyaluronic acid in combination with paclitaxel had significantly up-regulated the expression of vitamin D(3) binding proteins (DBP), which is a macrophage-stimulating activator. In conclusion, the in vivo anti-metastasis effect was associated with the synergistic therapeutic effects of hyaluronic acid-paclitaxel combination and a significant promotion of immune proteins expression. These results demonstrated that the combination of paclitaxel and hyaluronic acid may be an effective way to inhibit tumor migration.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Metástasis de la Neoplasia/prevención & control , Neoplasias del Cuello Uterino/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Ascitis/tratamiento farmacológico , Ascitis/patología , Proteínas Sanguíneas/análisis , Carcinoma Pulmonar de Lewis/sangre , Carcinoma Pulmonar de Lewis/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Electroforesis en Gel Bidimensional , Femenino , Ácido Hialurónico/administración & dosificación , Ácido Hialurónico/farmacología , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Metástasis de la Neoplasia/patología , Paclitaxel/administración & dosificación , Paclitaxel/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Análisis de Supervivencia , Porcinos , Resultado del Tratamiento , Neoplasias del Cuello Uterino/sangre , Neoplasias del Cuello Uterino/patologíaRESUMEN
Ce(4+) (Ce(NH(4))(2)(NO(3))(6)) at 1mM induces apoptosis of suspension cultures of Taxus cuspidata cells; however, the underlying signal mechanisms are unknown. We show here that a 46-kDa ERK (extracellular signal-regulated kinase)-like MAPK appears to be down-regulated at 4h, and remains at low levels for up to 48 h. An inhibitor of superoxide anions (O(2)(-)) generation, diphenyl iodonium (DPI) successfully blocks down-regulation of ERK-like MAPK and degradation of DNA. Moreover, a 41-kDa p38-like MAPK activity remains unchanged from 0.5 to 48 h. The p38 inhibitor SB202190 effectively inhibits p38-like MAPK activity, however, SB202190 fails to modify the apoptotic rate at concentrations up to 100 microM. Three nuclease (34-kDa, 22-kDa and 20-kDa) activities are profoundly enhanced in Ce(4+)-induced T. cuspidata cells. They have an optimum pH at 6.8, and are stimulated by Ca(2+)/Mg(2+). Caspase-3 inhibitor, Ac-DEVD-CHO, does not attenuate the 34-kDa nuclease activity, but inhibits the 22-kDa and the 20-kDa nuclease activities. In addition, inhibition of O(2)(-) generation by DPI significantly reduces the three nuclease activities. In conclusion, the present study suggests that down-regulation of ERK-like MAPK, burst of O(2)(-), activation of caspase-3-like and induction of three nucleases as the key signaling events mediating apoptosis in Ce(4+)-induced cultured T. cuspidata cells.
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Apoptosis/fisiología , Cerio/farmacología , Endonucleasas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Taxus/efectos de los fármacos , Taxus/enzimología , Apoptosis/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Endonucleasas/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Oxidantes/farmacología , Superóxidos/metabolismo , Superóxidos/farmacología , Taxus/citologíaRESUMEN
The signal events of 1 mM Ce4+ (Ce(NH4)2(NO3)6)-induced apoptosis of cultured Taxus cuspidata cells were investigated. The percentage of apoptotic cells increased from 0.82% to 51.32% within 6 days. Caspase-3-like protease activity became notable during the second day of Ce4+-treatment, and the maximum activity was 5-fold higher than that of control cells at the fourth day. When the experiment system was pretreated with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) at 100 microM, caspase-3-like activity resulted in distinct inhibition by 70% and 77.3% after 3 and 4 days of induction. Furthermore, 100 microM Ac-DEVD-CHO partially reduced the apoptotic cells by 58.6% and 60.8% at day 4 and 5 respectively. Ce4+ induced superoxide anions (O2*-) transient burst, and the first peak appeared at around 3.7-4 h, the second appeared at about 7 h. Both O2*- burst and cell apoptosis were effectively suppressed by application of diphenyl iodonium (NADPH oxidase inhibitor). Inhibition of O2*- production attenuated caspase-3-like activation by 49% and 53.6% during day 3 and 4 respectively. In addition, a total of 15 protein spots changed in response to caspase-3-like protease activation were identified by two-dimensional gel electrophoresis. These results suggest that Ce4+ of 1 mM induces apoptosis in suspension cultures of T. cuspidata through O2*- burst as well as caspase-3-like protease activation. The burst of O2*- exerts its activity as an upstream of caspase-3-like activation. Our results also implicate that other signal pathways independent of an O2*- burst possibly participate in mediating caspase-3-like protease activation.
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Apoptosis/efectos de los fármacos , Caspasas/clasificación , Caspasas/metabolismo , Cerio/farmacología , Transducción de Señal/efectos de los fármacos , Superóxidos/metabolismo , Taxus/citología , Caspasa 3 , Inhibidores de Caspasas , Cationes/farmacología , Línea Celular , Electroforesis en Gel Bidimensional , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacologíaRESUMEN
Two-liquid-phase plant cell cultures employ the use of a partitioning system to redirect extracellular product into a second phase. After the addition of organic solvent, in order to understand the defense system of Taxus cuspidata cells to organic solvent in two-liquid-phase suspension cultures, we investigated cells' antioxidant metabolism. The results showed that T. cuspidata cells responded to oleic acid with oxidative bursts in both intracellular H2O2 and extracellular O2-* production. Inhibition studies with diphenylene iodonium suggested that the key enzyme responsible for oxidative bursts was primarily NADPH oxidase. Investigation of the relationship between reactive oxygen species (ROS) and defense responses induced by oleic acid indicated that 4% (v/v) oleic acid increased the levels of antioxidant enzymes of superoxide dismutase, ascorbate peroxidase, and catalase and the antioxidant capacity of reduced ascorbate and glutathione. However, when oleic acid content reached a critical value (6% [v/v]), no further increase in antioxidant enzymes and antioxidant capacity was observed, indicating that the defense responses played a role in a certain range of oleic acid content, beyond which the overall ROS scavenging machinery was not induced and the peroxidation of membrane lipids emerged.
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Antioxidantes/metabolismo , Ácido Oléico/farmacología , Taxus/citología , Ascorbato Peroxidasas , Ácido Ascórbico/metabolismo , Catalasa/metabolismo , Células Cultivadas , Glutatión/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/análisis , NADPH Oxidasas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Peroxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Taxus/efectos de los fármacos , Taxus/metabolismo , Factores de TiempoRESUMEN
The interactions between sodium hyaluronate, an anionic polysaccharide, with surfactants (anionic and nonionic) were investigated using pyrene fluorescence measurement methods. The change of micropolarity produced by the interaction was monitored by the measurement of emission intensity ratio between the first and third bands (I1/I3), and the intensity ratio of the excimer and the third vibration monomer band (I(E)/I(M)). Because the hydrophilic heads on the SDS were attracted by the domains formed by the hydroxyl groups of hyaluronate, the I1/I3 ratio was reduced by the addition of hyaluronate at lower than 0.06% of sodium dodecyl sulfate (SDS) concentration. No aggregation was observed between hyaluronate and nonionic surfactants (Tween-80 and Cremophor EL) in the whole concentration range studied. At a higher concentration of surfactant, the I1/I3 ratio of hyaluronate/surfactant was influenced by the addition of saccharide (glucose, lactose, or mannitol). However, the effect of saccharide could be reduced by the addition of salt.
Asunto(s)
Ácido Hialurónico/química , Tensoactivos/química , Tampones (Química) , Polisorbatos/farmacología , Pirenos/química , Cloruro de Sodio/farmacología , Dodecil Sulfato de Sodio/farmacología , Soluciones/química , Espectrometría de FluorescenciaRESUMEN
The inclusion complexation behavior of paclitaxel with a series of oligo(ethylenediamino) bridged bis(beta-cyclodextrin)s possessing bridge chains in different length (1-4) has been investigated in order to improve the water solubility of paclitaxel. It is found that only the long-tethered bis(beta-cyclodextrin)s 1 and 2 can form the inclusion complexes with paclitaxel, which are characterized by NMR, SEM, XRD, FT-IR, TG-DTA, DSC, and microcalorimetry technology. The results obtained show that bis(beta-cyclodextrin)s 1 and 2 are able to solubilize paclitaxel to high levels up to 2 and 0.9 mg/mL, respectively. The high complex stability of bis(beta-cyclodextrin) 1 and paclitaxel is discussed from thermodynamic viewpoint. Furthermore, the cytotoxicity of these complexes assessed using a human erythroleukemia K562 cell line indicates that the IC(50) value of 1/paclitaxel complex is 6.0 x 10(-10) mol/dm(3) (calculated as paclitaxel molar concentration), which means that the antitumor activity of 1/paclitaxel complex is better than that of parent paclitaxel (IC(50) value 9.8 x 10(-10) mol/dm(3)). This high antitumor activity, along with the satisfactory water solubility and high thermal stability of the 1/paclitaxel complex, will be potentially useful for its clinical application as a highly effective antitumor drug.
Asunto(s)
Antineoplásicos/química , Paclitaxel/análogos & derivados , beta-Ciclodextrinas/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Humanos , Paclitaxel/farmacología , Solubilidad/efectos de los fármacos , beta-Ciclodextrinas/farmacologíaRESUMEN
Apoptosis-resistant K562 cells and apoptosis-proficient HL-60 acute myelomonocytic leukemia cells were selected to study the cell-type-specific characteristics of docetaxel. The kinetics of cytotoxicity of docetaxel showed a delayed response of K562 cells compared to HL-60 cells. After treatment with 10(-8)M docetaxel, DNA fragmentation and sub-G0/G1 cells were evident in HL-60 cells in less than 6 h, while K562 cells gradually arrested in G2/M phase of the cell cycle and appeared normal for 24 h before developing similar apoptotic changes. The delayed apoptotic changes in K562 cells were accompanied by delayed activation of caspase-3. Additionally, NADPH oxidase inhibition with diphenylene iodonium showed that reactive oxygen species (ROS) burst mediated critically in the caspase-3 activation and apoptosis in HL-60 cells but was only partially involved in those events of K562 cells. These results suggested that docetaxel exposure triggered the delayed apoptosis in K562 cells and the different ROS-dependent or independent signal pathways might account for this phenomenon. Docetaxel elicited ROS production from NADPH oxidase, which in turn triggered activation of caspase-3, leading to apoptosis in HL-60 cells. While in K562 cells, docetaxel induced apoptosis after G2/M accumulation through ROS-independent or partially dependent pathways.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/biosíntesis , Especies Reactivas de Oxígeno , Taxoides/farmacología , Caspasa 3 , Docetaxel , Inducción Enzimática , Células HL-60 , Humanos , Células K562 , CinéticaRESUMEN
The permeability of cell membrane was enhanced by exogenous SA in the culture of Taxus cuspidata. Nuclei condense and fragments were observed in some cells by using fluorescent microscope, and the degraded chromosomal DNA was observed by using agarose gel electrophoresis. The changes in soluble proteins of the suspension cultures of Taxus cuspidata induced by salicylic acids were analyzed by using two-dimensional polyacrylamide gel electrophoresis. Comparing with the control, seven new protein spots were found and six protein spots were not found in the cultures grown with SA at 48h. The results obtained showed that SA could induce the expression of some special proteins that might be related with the action of SA in the suspension cultures of Taxus cuspidata.