Ultrastructural aspects of melatonin cytotoxicity on Caco-2 cells in vitro.

Colon adenocarcinoma is a disease expanding worldwide. Cancer of colon and rectum are among the top ten most insidious types in Brazil. In vitro and in vivo studies have demonstrated the efficacy of the hormone melatonin to prevent and reduce tumor growth. However, there are only few studies addressing the action of melatonin on Caco-2 cells. Thus, the cytotoxic effect of melatonin on the ultrastructure of Caco-2 cells was investigated. The MTT colorimetric method was used to assess the cytotoxicity. A total of 2×10(6)cells/mL were seeded in microplates and incubated at 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0.0 (control) µg/mL of melatonin. For ultrastructural analysis concentrations with low, medium and high cytotoxicity plus the control were used for ultrastructural analysis. The concentrations 50, 1.56 and 0.78 µg/mL of melatonin showed low, medium and high cytotoxicity, respectively. Ultrastructurally, the control tumor cells were shown to be preserved. Caco-2 cells showed morphological changes at 50 µg/mL of melatonin, with numerous vacuoles, mitochondrial degeneration and reduced glycogen. However, Caco-2 cells also showed altered morphology in treatments at 1.56 and 0.78 µg/mL of melatonin with characteristics of cells in degeneration by the presence of numerous vacuoles, absence of microvilli, mitochondrial degeneration and nuclear fragmentation. Thus, one can infer that concentrations of 1.56 and 0.78 µg/mL of melatonin promote cytotoxicity in Caco-2 cells, which can probably be related to the generation of reactive oxygen species (ROS).

Kefiran antagonizes cytopathic effects of Bacillus cereus extracellular factors.

Kefiran, the polysaccharide produced by microorganisms present in kefir grains, is a water-soluble branched glucogalactan containing equal amounts of D-glucose and D-galactose. In this study, the effect of kefiran on the biological activity of Bacillus cereus strain B10502 extracellular factors was assessed by using cultured human enterocytes (Caco-2 cells) and human erythrocytes. In the presence of kefiran concentrations ranging from 300 to 1000 mg/L, the ability of B. cereus B10502 spent culture supernatants to detach and damage cultured human enterocytes was significantly abrogated. In addition, mitochondrial dehydrogenase activity was higher when kefiran was present during the cell toxicity assays. Protection was also demonstrated in hemolysis and apoptosis/necrosis assays. Scanning electron microscopy showed the protective effect of kefiran against structural cell damages produced by factors synthesized by B. cereus strain B10502. Protective effect of kefiran depended on strain of B. cereus. Our findings demonstrate the ability of kefiran to antagonize key events of B. cereus B10502 virulence. This property, although strain-specific, gives new perspectives for the role of bacterial exopolysaccharides in functional foods.

Influence of polarisation and differentiation on interaction of 43-kDa outer-membrane protein of Aeromonas caviae with human enterocyte-like Caco-2 cell line.

It has been recognised that adherence and invasion to host cells are important steps in the pathogenesis of entero-pathogenic bacteria, including Aeromonas caviae. However, the virulence factors of A. caviae remain, for the most part, poorly known. This study examined the interaction of A. caviae isolates to Caco-2 cells in different polarisation and differentiation conditions. The adherence of A. caviae may be related to accessibility of host cell basolateral receptors. Aggregative A. caviae isolates, grown at 22 degrees C, were more adherent in both non-polarised and undifferentiated Caco-2 cells and EGTA-treated polarised and differentiated Caco-2 cells. Furthermore, monolayers pre-incubated with 43-kDa outer-membrane protein (OMP) or A. caviae strains pre-incubated with rabbit IgG anti-43-kDa OMP decreased adherence of some A. caviae strains to EGTA-treated polarised and differentiated Caco-2 cells, suggesting an interaction of 43-kDa OMP with basolateral cell receptors. Bacterial cells were observed adhering to microvilli and to plasma membrane on both the apical and basal surfaces of the monolayer. Pedestal-like formation with cytoskeletal rearrangement was also observed. The bacteria entered the Caco-2 cells and were observed enclosed in single and multiple membrane-bound vacuoles within the host cell cytoplasm. Furthermore, A. caviae were observed free in the cytosol of Caco-2 cells, suggesting escape form cytoplasmatic vacuoles.

Effect of Bacillus cereus exocellular factors on human intestinal epithelial cells.

To gain insight on the biological effects of the exocellular factors produced by Bacillus cereus, culture filtrate supernatants of different strains were coincubated with differentiated Caco-2 cells. Exocellular factors were able to detach enterocyte-like cells from the substratum after 1 h of incubation. In addition, microvilli effacing and dramatic changes on the cellular surface of enterocytes were found after incubation periods as short as 20 min. Since cell detachment was not inhibited by fetal calf serum, thiol activated cholesterol-binding cytolysin, cereolysin O, does not seem to be involved. Also, translocation of phosphatidylserine from the inner to the outer leaflets of the plasma membrane was demonstrated by using fluorescein isothiocyanate (FITC)-Annexin V. In contrast to the high capability of detaching Caco-2 cells shown by all the strains under study, the mitochondrial dehydrogenase activity was lowered by culture filtrate supernatants in a strain-dependent manner. For strain M2, the decrease in dehydrogenase activity was already evident after 30 min of incubation. Production of biologically active factors depends on the growth phase, and maximal activity was found in late exponential-early stationary phases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of concentrated exocellular factors showed a very complex scenery supporting the multifactorial character of the biological activity of B. cereus.