RESUMEN
Ginsenoside Rh2 increases the efficacy of doxorubicin (DOX) treatment in murine models of solid and ascites Ehrlich's adenocarcinoma. In a solid tumor model (treatment commencing 7 days after inoculation), DOX + Rh2 co-treatment was significantly more efficacious than DOX alone. If treatment was started 24 h after inoculation, the inhibition of tumor growth of a solid tumor for the DOX + Rh2 co-treatment group was complete. Furthermore, survival in the ascites model was dramatically higher for the DOX + Rh2 co-treatment group than for DOX alone. Mechanisms underlying the combined DOX and Rh2 effects were studied in primary Ehrlich's adenocarcinoma-derived cells and healthy mice's splenocytes. Despite the previously established Rh2 pro-oxidant activity, DOX + Rh2 co-treatment revealed no increase in ROS compared to DOX treatment alone. However, DOX + Rh2 treatment was more effective in suppressing Ehrlich adenocarcinoma cell adhesion than either treatment alone. We hypothesize that the benefits of DOX + Rh2 combination treatment are due to the suppression of tumor cell attachment/invasion that might be effective in preventing metastatic spread of tumor cells. Ginsenoside Rh2 was found to be a modest activator in a Neh2-luc reporter assay, suggesting that Rh2 can activate the Nrf2-driven antioxidant program. Rh2-induced direct activation of Nrf2 might provide additional benefits by minimizing DOX toxicity towards non-cancerous cells.
Asunto(s)
Adenocarcinoma , Medicamentos Herbarios Chinos , Ginsenósidos , Animales , Doxorrubicina/farmacología , Medicamentos Herbarios Chinos/farmacología , Ginsenósidos/farmacología , RatonesRESUMEN
The tubular immunostimulating complex (TI-complex) consisting of cucumarioside A2-2, cholesterol and monogalactosyldiacylglycerol (MGDG) from marine macrophytes is the perspective antigen delivery system for subunit vaccines. MGDG is a lipid matrix for the protein antigen incorporated in the TI-complex. The aim of the present work was to study the influence of MGDGs from different macrophytes on conformation and immunogenicity of the secreted recombinant uncleaved hemagglutinin monomer (HA0S) of influenza A virus H1/N1. Differential scanning calorimetry, fluorescence spectroscopy and circular dichroism showed a dependence of the conformational changes of HA0S on the microviscosity of MGDG. The most viscous MGDG from Zostera marina induced the strongest rearrangements in protein conformation. Immunization of mice with HA0S within TI-complexes comprising different MGDGs resulted in an approximately 2-fold increase of the levels of anti-HA0S antibodies and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared with those induced by HA0S alone. TI-complexes based on MGDG from Z. marina stimulated the maximal production of GM-CSF. However, humoral immune response (anti-HA0S antibodies), unlike cell-mediated immune response (GM-CSF), did not depend on the physicochemical properties of MGDGs. It is assumed that this is due to the different localization and conformational lipid sensitivity of the HA0S regions, which are responsible for these types of immune responses.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos Virales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Liposomas/farmacología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/aislamiento & purificación , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/química , Calorimetría , Dicroismo Circular , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Vacunas contra la Influenza/química , Liposomas/química , Liposomas/aislamiento & purificación , Ratones , Conformación Proteica , Espectrometría de Fluorescencia , Vacunación/métodos , Vacunas de Subunidad/química , Vacunas de Subunidad/inmunología , Zosteraceae/químicaRESUMEN
The tubular immunostimulating complex (TI-complex) is a novel nanoparticulate antigen delivery system consisting of cholesterol, triterpene glycoside cucumarioside A(2)-2, and glycolipid monogalactosyldiacylglycerol (MGDG) isolated from marine macrophytes. MGDG is crucial for the formation of a lipid matrix for the protein antigen incorporated in TI-complexes. Fatty acid composition and the physical state of this glycolipid depend on the taxonomic position of marine macrophytes. Therefore, the aim of the present work was to study the capacity of MGDGs, isolated from five species of marine macrophytes, to influence conformation and to enhance immunogenicity of porin from Yersinia pseudotuberculosis (YOmpF) as a model antigen of subunit vaccine based on TI-complexes. The trimeric porin was chosen for these experiments, because it was approximately two times more immunogenic than monomeric porin incorporated in TI-complexes. Immunization of mice with YOmpF within TI-complexes, comprised of different MGDGs, revealed a dependence of the immunostimulating effect of TI-complexes on the microvicosity of this glycolipid. TI-complexes comprising MGDGs from Sargassum pallidum and Ulva fenestrata with medium microviscosity induced maximal levels of anti-porin antibodies (four times higher when compared with those induced by pure porin). The adjuvant effect of TI-complexes based on other MGDGs varied by 2.8, 2.3 and 1.3 times for TI-complexes comprised of MGDGs from Zostera marina, Ahnfeltia tobuchiensis, and Laminaria japonica, respectively. MGDGs are also able to influence cytokine mechanisms of immunological regulation. DSC and spectroscopic studies showed that maximal immunostimulating effect of TI-complexes correlated with a moderate stabilizing influence of MGDGs from S. pallidum and U. fenestrata on the conformation of porin. The results obtained suggest lipid "nanofluidics" as a novel strategy for optimizing the immune response to protein antigens within lipid particulate systems.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos Bacterianos/inmunología , Galactolípidos/farmacología , Extractos Vegetales/farmacología , Porinas/inmunología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/aislamiento & purificación , Algoritmos , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/química , Rastreo Diferencial de Calorimetría , Citocinas/sangre , Ácidos Grasos/química , Femenino , Galactolípidos/química , Galactolípidos/aislamiento & purificación , Inmunización , Laminaria/química , Ratones , Ratones Endogámicos BALB C , Nanopartículas , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Porinas/química , Estructura Secundaria de Proteína , Rhodophyta/química , Sargassum/química , Espectrometría de Fluorescencia , Ulva/química , Viscosidad , Yersinia pseudotuberculosis , Zosteraceae/químicaRESUMEN
BACKGROUND: There is an urgent need to develop safe and effective adjuvants for the new generation of subunit vaccines. We developed the tubular immunostimulating complex (TI-complex) as a new nanoparticulate antigen delivery system. The morphology and composition of TI-complexes principally differ from the known vesicular immunostimulating complexes (ISCOMs). However, methodology for the preparation of TI-complexes has suffered a number of shortcomings. The aim of the present work was to obtain an antigen carrier consisting of triterpene glycosides from Cucumaria japonica, cholesterol, and monogalactosyldiacylglycerol from marine macrophytes with reproducible properties and high adjuvant activity. RESULTS: The cucumarioside A2-2 - cholesterol - MGalDG ratio of 6:2:4 (by weight) was found to provide the most effective formation of TI-complexes and the minimum hemolytic activity in vitro. Tubules of TI-complexes have an outer diameter of about 16 nm, an inner diameter of 6 nm, and a length of 500 nm. A significant dilution by the buffer gradually destroyed the tubular nanoparticles. The TI-complex was able to increase the immunogenicity of the protein antigens from Yersinia pseudotuberculosis by three to four times. CONCLUSIONS: We propose an optimized methodology for the preparation of homogeneous TI-complexes containing only tubular particles, which would achieve reproducible immunization results. We suggest that the elaborated TI-complexes apply as a universal delivery system for different subunit antigens within anti-infectious vaccines and enhance their economic efficacy and safety.