RESUMEN
Nanotechnology is providing new tools for precision agriculture, such as agrochemical agents and innovative delivery mechanisms to improve cropping efficiency. Powder nanoinsecticides, such as experimental nanostructured alumina (NSA), show great potential for sustainable agriculture as an alternative to conventional synthetic pesticides because their mechanism of insecticide action is based on physical rather than on biochemical phenomena. However, even in highly non-reactive and hardly soluble substances such as alumina, reduced particle size may lead to an increased toxicity of the material. In order to determine whether NSA induces DNA and chromosomal damage, its toxicity was assessed in human peripheral blood lymphocytes (PBL) and contrasted with commercial nanostructured alumina, natural insecticide powders and a conventional pesticide. PBL from healthy donors were exposed for 24 h to increasing concentrations (50, 100 and 200 µg/mL) of NSA particle agglomerates (<350 nm); positive and negative NSA-particles, respectively; bulk Al2O3 (4.5 µm) or Diatomaceous Earth (SiO2, <4.5 µm). Alkaline comet assay and micronuclei (MNi) test were used to assess DNA damage and chromosomal breakage, respectively. Cell viability was tested with resazurin assay. Comet assay results revealed no significant increase in DNA damage by NSA compared to other natural substances. As expected, DNA breaks were significantly higher in cells exposed to an organophosphate [OPP] control (P < 0.05). No statistically significant differences were found in terms of cellular viability at 50 and 100 µg/mL of NSA but cell survival decreased at 200 µg/mL as well as in OPP group. Positively charged NSA particles significantly reduced cell viability and increased DNA migration and oxidative DNA damage (8-oxoG). NSA as well as the electrically charged NSA particles had no significant effect on MNi induction. Our results indicate that NSA particles are non-cytotoxic and non-genotoxic at the tested doses and do not cause obvious DNA damage in human PBL in vitro.
RESUMEN
Heat shock proteins (Hsp) are molecular chaperones with the capability to interact with a wide range of other proteins and are thus often found coupled with other heat shock and non-heat shock proteins. This can be an advantage to study specific interactions between a chaperone and other proteins and to generate an antitumoral immune response. In this chapter, we present two protocols to isolate Hsp. One involves column chromatography with hydroxyapatite and the other employs immunoprecipitation with antibodies coupled to magnetic beads. In both cases, we specifically want to isolate Hsp coupled with other proteins and use the Hsp complexes as intermediaries to present the coupled peptides/proteins to the immune system, or to explore the associations of a particular Hsp with other proteins.
Asunto(s)
Cromatografía de Afinidad/métodos , Proteínas de Choque Térmico/aislamiento & purificación , Inmunoprecipitación/métodos , Materiales Biocompatibles , Línea Celular Tumoral , Durapatita , Proteínas de Choque Térmico/química , Humanos , Dominios y Motivos de Interacción de ProteínasRESUMEN
The heat shock proteins (HSP) constitute a superfamily of chaperone proteins present in all cells and in all cell compartments, operating in a complex interplay with synergistic/overlapping multiplicity of functions, even though the common effect is cell protection. Several reasons explain the need for investigating HSP in prostate cancer: (1) these molecules function as chaperones of tumorigenesis accompanying the emergence of prostate cancer cells, (2) they appear as useful molecular markers associated with disease aggressiveness and with resistance to anticancer therapies including hormone therapy, radiotherapy, chemotherapy and hyperthermia, and (3) they can be used as targets for therapies. The latter can be accomplished by: (i) interrupting the interaction of HSP (mainly HSPC1) with various client proteins that are protected from degradation when chaperoned by the HSP; (ii) using the chaperone and adjuvant capabilities of certain HSP to present antigenic peptides to the immune system, so this system can recognise the prostate tumour cells as foreign to mount an effective antitumoral response; and (iii) using treatment planning models taking into account the HSP expression levels to obtain more effective therapies. In summary, the study of the HSP during tumorigenesis as well as during cancer progression, and the inclusion of treatment designs targeting HSP combined with other treatment modalities, should improve prostate cancer survival in the near future.