Investigation of apoptosis in cultured cells infected with equine herpesvirus 1.

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin-Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.

Microvascular lesions and changes in cell proliferation and death, and cytokine expression in the placentas of mice experimentally infected with Equid Herpesvirus 1.

This study describes the changes observed in the placentas of mice experimentally infected with an abortigenic strain of EHV-1 at mid-pregnancy and euthanized at days 3 and 4 post-infection. We analyzed microscopic vascular alterations, cell proliferation and death by immunohistochemistry, and the expression of IFN-γ, TNF-α and the IL-10 by qPCR and flow cytometry. Infected mice showed slight respiratory signs and ruffled fur during the first two days post-infection. Virus isolation and DNA detection were positive only in the lungs of the infected mice. Vascular congestion, increase in the labyrinth area, and a significant reduction in fetal capillary endothelium surface of infected placentas were found. Cell proliferation was significantly reduced in the infected placentas, whereas the apoptosis was significantly increased. IL10, TNF and IFN-γ showed different expression in the infected placentas and uteri. The effects of EHV-1 during pregnancy depend on different pathogenic mechanisms in which vascular alterations, and cell death and proliferation and local cytokine changes are compromised.

Vanadium and cancer treatment: antitumoral mechanisms of three oxidovanadium(IV) complexes on a human osteosarcoma cell line.

We report herein the antitumor actions of three oxidovanadium(IV) complexes on MG-63 human osteosarcoma cell line. The three complexes: VO(oda), VO(oda)bipy and VO(oda)phen (oda=oxodiacetate), caused a concentration dependent inhibition of cell viability. The antiproliferative action of VO(oda)phen could be observed in the whole range of concentrations (at 2.5 µM), while VO(oda)bipy and VO(oda) showed a decrease of cell viability only at higher concentrations (at 50 and 75 µM, respectively) (p<0.01). Moreover, VO(oda)phen caused a decrease of lysosomal and mitochondrial activities at 2.5 µM, while VO(oda) and VO(oda)bipy affected neutral red uptake and mitochondrial metabolism at 50 µM (p<0.01). On the other hand, no DNA damage studied by the Comet assay could be observed in MG-63 cells treated with VO(oda) at 2.5-10 µM. Nevertheless, VO(oda)phen and VO(oda)bipy induced DNA damage at 2.5 and 10 µM, respectively (p<0.01). The generation of reactive oxygen species increased at 10 µM of VO(oda)phen and only at 100 µM of VO(oda) and VO(oda)bipy (p<0.01). Besides, VO(oda)phen and VO(oda)bipy triggered apoptosis as determined by externalization of the phosphatidylserine. The determination of DNA cleavage by agarose gel electrophoresis showed that the ability of VO(oda)(bipy) is similar to that of VO(oda), while VO(oda)(phen) showed the highest nuclease activity in this series. Overall, our results showed a good relationship between the bioactivity of the complexes and their structures since VO(oda)phen presented the most potent antitumor action in human osteosarcoma cells followed by VO(oda)bipy and then by VO(oda) according to the number of intercalating heterocyclic moieties.

Antitumor properties of a vanadyl(IV) complex with the flavonoid chrysin [VO(chrysin)2EtOH]2 in a human osteosarcoma model: the role of oxidative stress and apoptosis.

Flavonoids, a polyphenolic compound family, and the vanadium compounds have interesting biological, pharmacological, and medicinal properties. We report herein the antitumor actions of the complex [VO(chrysin)2EtOH]2 (VOchrys) on the MG-63 human osteosarcoma cell line. Oxovanadium(IV), chrysin and VOchrys caused a concentration-dependent inhibition of cell viability. The complex was the strongest antiproliferative agent (p < 0.05). Cytotoxicity and genotoxicity studies also showed a concentration effect. Reactive oxygen species (ROS) and the alterations in the GSH/GSSG ratio underlie the main mechanisms of action of VOchrys. Additions of ROS scavengers (vitamin C plus vitamin E) or GSH to the viability experiments demonstrated beneficial effects (p < 0.01). Besides, the complex triggered apoptosis, disruption of the mitochondria membrane potential (MMP), increased levels of caspase 3 and DNA fragmentation measured by the sub-G1 peak in cell cycle arrest experiments (p < 0.01). Collectively, VOchrys is a cell death modulator and a promissory complex to be used in cancer treatments.

Cytotoxicity of a vanadyl(IV) complex with a multidentate oxygen donor in osteoblast cell lines in culture.

Strong chelating ligands as oxodiacetate (oda) are model systems to study the process of metal trapping by living organisms. Vanadium compounds display interesting biological and pharmacological actions. In vertebrates, vanadium is stored mainly in bones. In the present study we report the effects of the complex of oda with vanadyl(IV) cation, VO(oda), on two osteoblast cell lines, one normal (MC3T3E1) and the other tumoral (UMR106). VO(oda) exerted cytotoxic actions in osteoblasts as it was determined through a dose-dependent decrease in cell proliferation, and morphological and actin alterations. The putative mechanisms underlying VO(oda) deleterious effects were also investigated. The complex increased the level of ROS which correlated with a decreased in GSH/GSSG ratio. Besides, VO(oda) induced a dissipation of the mitochondria membrane potential (MMP) and promoted an increase in ERK cascade phosphorylation, which is involved in the regulation of cellular death and survival. All the effects were more pronounced in MC3T3-E1 than in UMR106 cells. ERK activation was inhibited by PD98059, Wortmanin and the ROS scavenger NAC (N-acetyl cysteine). These results suggest that VO(oda) stimulated ERKs phosphorilation by induction of free radicals involving kinases upstream of ERK pathway. The inhibitory effect of the complex on cell proliferation was partially reversed in both cell lines by NAC. Moreover, PD98059 and Wortmanin also partially reversed the inhibition of cell proliferation in the tumoral osteoblasts. The use of specific inhibitors and ROS scavengers suggested the involvement of oxidative stress, MMP alterations and ERK pathway in the apoptotic actions of this complex.

Glutathione is involved in environmental stress responses in Rhizobium tropici, including acid tolerance.

The isolation of rhizobial strains which exhibit an intrinsic tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance. Rhizobium tropici strain CIAT899 displays a high intrinsic tolerance to acidity and therefore was used in this work to study the molecular basis of bacterial responses to acid conditions and other environmental stresses. We generated a collection of R. tropici CIAT899 mutants affected in acid tolerance using Tn5-luxAB mutagenesis, and one mutant strain (CIAT899-13T2), which fails to grow under acid conditions, was characterized in detail. Strain CIAT899-13T2 was found to contain a single Tn5-luxAB insertion in a gene showing a high degree of similarity with the Escherichia coli gshB gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and intracellular pH levels were found to be lower in the mutant than in the parent. The glutathione-deficient mutant was shown to be sensitive to weak organic acids, osmotic and oxidative stresses, and the presence of methylglyoxal. Glutathione restores responses to these stresses almost to wild-type levels. Our data show that in R. tropici the production of glutathione is essential for growth in extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by the wild-type strain in coinoculation experiments.