RESUMEN
Cyanobacteria produce many biologically active metabolites synthesized via nonribosomal synthetic pathways such as cyclic microcystins (MCs) and linear aeruginosins (Aers). The present study aimed to investigate the effects of different MC variants and the newly isolated aerugenosin Aer-865 on macrophages, which represent one of the key effector cells within the innate immune responses. Specifically, our study included RAW 264.7 macrophage activation associated with production of cytotoxic and cytostatic nitric oxide (NO) as well as pro-inflammatory mediators like tumor necrosis factor α (TNFα) and interleukin 6 (IL-6). From the compounds investigated, commonly occurring MC variants (-RR, -YR) and Aer-865 had no significant effects within the non-cytotoxic concentrations tested, i.e. 0.001-1⯵M for MCs and 0.1-50⯵M for Aer-865. In contrast to known immunoactive MC-LR, the negligible immunomodulatory potential of tested MC congeners could be related to their differences in structure. The knowledge of MC structure-specific activities contributes to the understanding of complex toxicity of different MC variants and most importantly their mixtures. This study is one of the first study that evaluate the effect of larger set of cyanobacterial peptides on macrophages and compare their immunomodulatory potential.
Asunto(s)
Toxinas Bacterianas/toxicidad , Bacteriocinas/toxicidad , Inmunomodulación/efectos de los fármacos , Macrófagos/efectos de los fármacos , Toxinas Marinas/toxicidad , Microcistinas/toxicidad , Animales , Toxinas Bacterianas/química , Bacteriocinas/química , Toxinas de Cianobacterias , Macrófagos/inmunología , Toxinas Marinas/química , Ratones , Microcistinas/química , Células RAW 264.7RESUMEN
The production of cytotoxic molecules interfering with mammalian cells is extensively reported in cyanobacteria. These compounds may have a use in pharmacological applications; however, their potential toxicity needs to be considered. We performed cytotoxicity tests of crude cyanobacterial extracts in six cell models in order to address the frequency of cyanobacterial cytotoxicity to human cells and the level of specificity to a particular cell line. A set of more than 100 cyanobacterial crude extracts isolated from soil habitats (mainly genera Nostoc and Tolypothrix) was tested by MTT test for in vitro toxicity on the hepatic and non-hepatic human cell lines HepG2 and HeLa, and three cell systems of rodent origin: Yac-1, Sp-2 and Balb/c 3T3 fibroblasts. Furthermore, a subset of the extracts was assessed for cytotoxicity against primary cultures of human hepatocytes as a model for evaluating potential hepatotoxicity. Roughly one third of cyanobacterial extracts caused cytotoxic effects (i.e. viability<75%) on human cell lines. Despite the sensitivity differences, high correlation coefficients among the inhibition values were obtained for particular cell systems. This suggests a prevailing general cytotoxic effect of extracts and their constituents. The non-transformed immortalized fibroblasts (Balb/c 3T3) and hepatic cancer line HepG2 exhibited good correlations with primary cultures of human hepatocytes. The presence of cytotoxic fractions in strongly cytotoxic extracts was confirmed by an activity-guided HPLC fractionation, and it was demonstrated that cyanobacterial cytotoxicity is caused by a mixture of components with similar hydrophobic/hydrophilic properties. The data presented here could be used in further research into in vitro testing based on human models for the toxicological monitoring of complex cyanobacterial samples.
Asunto(s)
Mezclas Complejas/toxicidad , Cianobacterias/química , Citotoxinas/análisis , Animales , Células 3T3 BALB , Línea Celular , Fibroblastos , Células HeLa , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Ratones , Sales de Tetrazolio , TiazolesRESUMEN
Aeruginosin-865 (Aer-865), isolated from terrestrial cyanobacterium Nostoc sp. Lukesová 30/93, is the first aeruginosin-type peptide containing both a fatty acid and a carbohydrate moiety, and is the first aeruginosin to be found in the genus Nostoc. Mass spectrometry, chemical and spectroscopic analysis as well as one- and two-dimensional NMR and chiral HPLC analysis of Marfey derivatives were applied to determine the peptidic sequence: D-Hpla, D-Leu, 5-OH-Choi, Agma, with hexanoic and mannopyranosyl uronic acid moieties linked to Choi. We used an AlphaLISA assay to measure the levels of proinflammatory mediators IL-8 and ICAM-1 in hTNF-α-stimulated HLMVECs. Aer-865 showed significant reduction of both: with EC50 values of (3.5±1.5) µg mL(-1) ((4.0±1.7) µM) and (50.0±13.4) µg mL(-1) ((57.8±15.5) µM), respectively. Confocal laser scanning microscopy revealed that the anti-inflammatory effect of Aer-865 was directly associated with inhibition of NF-κB translocation to the nucleus. Moreover, Aer-865 did not show any cytotoxic effect.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Glicopéptidos/farmacología , Lipopéptidos/farmacología , Nostoc/química , Transporte Activo de Núcleo Celular/efectos de los fármacos , Antiinflamatorios no Esteroideos/aislamiento & purificación , Línea Celular , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Glicopéptidos/química , Glicopéptidos/aislamiento & purificación , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-8/biosíntesis , Lipopéptidos/química , Lipopéptidos/aislamiento & purificación , Estructura Molecular , FN-kappa B/metabolismo , Nostoc/crecimiento & desarrollo , Transporte de Proteínas/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Chronic inflammation is at least partially mediated by the chemokine-mediated attraction and by the adhesion molecule-directed binding of leukocytes to the activated endothelium. Therefore, it is therapeutically important to identify anti-inflammatory compounds able to control the interaction between leukocytes and the endothelial compartments of the micro- and macrocirculation. When testing novel drug candidates, it is, however, of the utmost importance to detect side effects, such as potential cytotoxic and barrier-disruptive activities. Indeed, minor changes in the endothelial monolayer integrity may increase the permeability of small blood vessels and capillaries, which, in extreme cases, can lead to edema development. Here, we describe the development of a high-throughput screening (HTS) platform, based on AlphaLISA technology, able to identify anti-inflammatory nontoxic natural or synthetic compounds capable of reducing tumor necrosis factor (TNF)-induced chemokine (interleukin [IL]-8) and adhesion molecule (ICAM-1) expression in human lung microvascular endothelial cells. Quantification of cell membrane-expressed ICAM-1 and of cell culture supernatant-associated levels of IL-8 was analyzed in HTS. In parallel, we monitored monolayer integrity and endothelial cell viability using the electrical cell substrate impedance sensing method. This platform allowed us to identify natural secondary metabolites from cyanobacteria, capable of reducing ICAM-1 and IL-8 levels in TNF-activated human microvascular endothelial cells in the absence of endothelial monolayer barrier disruption.