Combination Of Cytochalasin Research Articles

Combination of Cytochalasin H and zinc oxide nanoparticles in human breast cancer: an insight into apoptosis study

Nanoparticle-mediated combination therapies have significant impact on improving the therapeutic efficacy of cancer. The aim of the current work was to investigate the apoptotic efficacy of Zinc Oxid nanoparticles (ZnONPs) in combination with Cytochalasin H (CytoH) in human MCF-7 breast cancer cells. The anti cancer activity of combined treatment of CytoH and ZnONPs was performed using MTT assay, Annexin V-propidium iodide staining, Caspase assay, and Hoechst 33342 staining. Quantitative real-time polymerase chain reaction was conducted to detect of p53 as a tumor suppressor gene expression in cancerous cells. Also, measurement of ROS generation was performed using flow cytometry. According to the MTT assay results, the combination of CytoH and ZnONPs significantly decreased the viability of cancer MCF-7 cells than either CytoH or ZnONPs alone as compared with normal HEK293 cells. The experiments, such as caspase 8 and 9 assay and Hoechst 33342 staining, showed that the combined treatment of CytoH and ZnONPs significantly increased the level of cell apoptosis pathway as compared with breast cancer cells treated with CytoH or ZnONPs alone. As demonstrated by annexin V/PI detection assay, the percentage of apoptotic MCF-7 cells was significantly increased, following combined treatment with CytoH and ZnONPs as compared with CytoH or ZnONPs alone. Moreover, by comparing the combination of CytoH and ZnONPs to CytoH and ZnONPs alone the ability of combined treatment to up-regulate p53 gene expression was observed. The results of the present study showed that combination of CytoH and ZnONPs could have a powerful synergistic cytotoxic effect on breast cancer cell line. Moreover, this is the first report indicating a new insight into how CytoH and ZnONPs can enhance apoptosis and cell growth in breast cancer cells.

Sevoflurane modulates the release of reactive oxygen species, myeloperoxidase, and elastase in human whole blood: Effects of different stimuli on neutrophil response to volatile anesthetic in vitro.

Volatile anesthetics have been shown to modulate polymorphonuclear neutrophil (PMN) functions. The aim of this study was to examine the impact of clinically relevant concentrations of sevoflurane (SEVO), a volatile anesthetic, on the release of reactive oxygen species (ROS), myeloperoxidase (MPO), and elastase (EL) from human activated PMNs. For this purpose, samples of whole blood were collected from healthy volunteers and exposed in vitro to 2.3% or 4.6% SEVO in air. To assess for a stimulus-dependent effect of the volatile anesthetic, PMNs were activated using different validated protocols. Artificial stimulation of neutrophils involved either a combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). In addition, a combination of lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) was also tested as a natural activation mean of PMNs. The production of ROS by PMNs was assessed by L-012 chemiluminescence. Total MPO and EL released in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, degranulation of the active fraction of MPO was also measured by specific immunological extraction followed by enzymatic detection (SIEFED). Overall, SEVO enhanced the release of ROS, MPO, and EL following artificial stimulation of PMNs but the volatile anesthetic inhibited the degranulation of active MPO and EL after neutrophil exposure to LPS and TNF-α. This study highlighted that the effect of SEVO on activated PMNs is dependent on the conditions of cell stimulation. These properties should be taken into consideration in future studies investigating immunomodulatory effects of volatile anesthetics.

Sevoflurane inhibits equine myeloperoxidase release and activity in vitro

ObjectiveTo investigate the effects of the volatile anaesthetic sevoflurane on the release of total and active myeloperoxidase (MPO) by non-stimulated and stimulated polymorphonuclear neutrophils (PMNs) in whole blood from healthy horses. Study designIn vitro experimental study. AnimalsAdult healthy horses. MethodsSamples of whole venous blood were collected and incubated in air or in air plus 2.3% or 4.6% sevoflurane for 1 hour. PMNs were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), with a combination of cytochalasin B (CB) and fMLP or with phorbol myristate acetate (PMA). Total and active MPO contents released by PMNs in blood were measured by enzyme-linked immunosorbent assay (ELISA) and specific immunological extraction followed by enzymatic detection (SIEFED) respectively. Additional experiments were performed to assess the effect of sevoflurane on the peroxidase and chlorination cycles of purified equine MPO using Amplex Red and 3’-(p-aminophenyl) fluorescein as fluorogenic substrates respectively. ResultsAs compared with air alone, 1 hour exposure of whole blood to 4.6% sevoflurane in air significantly inhibited the release of total and active MPO by unstimulated and both fMLP- and CB + fMLP-stimulated PMNs but not by PMA-stimulated PMNs. Although 2.3% sevoflurane had no effect on total MPO release by unstimulated and stimulated PMNs, it significantly reduced the release of active MPO by unstimulated and fMLP-stimulated PMNs. Additionally, sevoflurane reversibly inhibited the activity of MPO, especially the peroxidase cycle of the enzyme. Conclusions and clinical relevanceAlthough our experimental study was not designed to assess the effects of sevoflurane in vivo, this inhibition of MPO release and activity may have relevance for anaesthetized horses and deserves further studies to examine the clinical importance of these findings.

An in vitro whole blood model to test the effects of different stimuli conditions on the release of myeloperoxidase and elastase by equine neutrophils

Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate equine neutrophils in whole blood and to evaluate their response by measuring the release of total and active myeloperoxidase (MPO) and total elastase, considered as markers of neutrophil stimulation and degranulation. Because of the critical importance of the concomitant presence of LPS and TNF-α in equine pathological situations, we combined these two natural mediators to stimulate PMN and compared the response with those obtained after the PMN stimulation with each mediator used alone and well-known artificial stimulation systems such as 12-phorbol 13-myristate acetate (PMA) and the combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP). All the activation systems, PMA, CB/fMLP, TNF-α, LPS and LPS/TNF-α, induced a significant release of total MPO in whole blood but only the combinations CB/fMLP and LPS/TNF-α significantly favored the release of active MPO. Regarding the total elastase, we did not observe a significant release in all the stimulated conditions except with PMA. It appears clearly that the choice of the neutrophil stimulation model is fundamental for the selection of potentially active pharmacological agents, especially on MPO activity.

53 THE EFFECT OF ELECTRICAL PULSE ON DEVELOPMENT OF NT BOVINE EMBRYOS FROM CARTILAGE CELLS

In some previous NT studies, it was reported that oocyte activation starts with electrical stimulation applied before chemical activation (Rzucidlo SJ et al. 2004 Reprod. Fertil. Dev. 16(1,2), 157; Arat S et al. 2006 Reprod. Fertil. Dev. 18(1,2), 119). Therefore; one of the several parameters affecting reprogramming of somatic cells is the fusion parameter and time. Electrical stimulation has also an effect on embryo quality (Milazzotto MP et al. 2008 Reprod. Dom. Anim. 43, 319–322). The objective of this study was to examine the effect of fusion parameter and time on blastocysts development rates on somatic cell nucleus transfer(SCNT). Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with 10% fetal bovine serum (FBS), sodiumpyruvate, penicillin/streptomycin, epidermal growth factor (EGF), bFSH, and bLH in a humidified atmosphere of 5% CO2 in air for 18 h. Single cells derived from cartilage tissue of Anatolian Black Cow were inserted into the perivitelline space of the enucleated oocytes. In the first experiment, NT couples were fused by 2.66 kV cm–1, 30 μs, 1 pulse (Group 1) in sorbitol fusion buffer (0.25 m sorbitol, 0.1 mm Calcium acetate, 0.5 mm Mg Acetate). After one hour of the first fusion nonfused NT couples were refused by 1.40 kV cm–1, 40 μs, 1 pulse (Group 2). After fusion, all fused NT couples in group 1 (1.5 h after the first fusion) and 2 (1.5 h after the second fusion) were activated using a combination of cytochalasin D (2.5 μg mL–1) and cycloheximide (CHX, 10 μg mL–1) for 1 h and CHX alone for 4 h. Following activation, reconstructed oocytes were cultured in Sage cleavage medium supplemented with 8 mg mL–1 BSA for 72 h and then developing embryos were cultured in Sage blastocyst media (Tang R et al. 2006 Human Reproduction 21(5), 1179–1183) supplemented with 4 mg mL–1 BSA + 5% FCS for an additional 4 days. Differences among groups were analyzed by one-way ANOVA after arcsin square transformation(P = 0.05). There was no significant difference on fusion rates observed between Group 1(49.4%) and Group 2(47.5%). However, the result showed that the blastocyst development was seriously decreased after second fusion(Group 1: 30.8% and group 2: 2.5%). This was considered as a negative effect of the electrical stimulation which was applied twice on NT couples. In the second experiment; development rates of embryos were compared from NT couples fused 24 or 28 hours after maturation. In this experiment, fusion was applied one time. NT couples fused approximately 24 and 28 h postmaturation were considered as early and late, respectively. The results showed that there was no significant differences between the two groups on the blastocysts development rates (31.6% early and 28.4% late). According to the these results, 4 h difference at oocyte ages was not an effective parameter on the blastocyst development rates of NT embryos. This study was supported by a grant from TUBITAK TOVAG 104O360 and KAMAG, Turkey (106G005).

A Novel Method for Somatic Cell Nuclear Transfer to Mouse Embryonic Stem Cells

Nuclear reprogramming by somatic cell nuclear transfer (SCNT) provides a practical approach for generating autologous pluripotent cells from adult somatic cells. It has been shown that murine somatic cells can also be reprogrammed to a pluripotent-like state by fusion with embryonic stem (ES) cells. Typically, the first step in SCNT involves enucleation of the recipient cell. However, recent evidence suggests that enucleated diploid ES cells may lack reprogramming capabilities. Here we have developed methods whereby larger tetraploid ES cells are first generated by fusion of two mouse ES cell lines transfected with plasmids carrying different antibiotic-resistance cassettes, followed by double antibiotic selection. Tetraploid ES cells grown on tissue culture disks or wells can be efficiently enucleated (up to 99%) using a combination of cytochalasin B treatment and centrifugation, with cytoplasts generated from these cells larger than those obtained from normal diploid ES cells. Also, we show that the enucleation rate is dependent on centrifugation time and cell ploidy. Further, we demonstrate that normal diploid ES cells can be fused to tetraploid ES cells to form heterokaryons, and that selective differential centrifugation conditions can be applied where the tetraploid nucleus is removed while the diploid donor nucleus is retained. This technology opens new avenues for generating autologous, diploid pluripotent cells, and provides a dynamic model for studying nuclear reprogramming in ES cells.

Hypertonicity-induced projections reflect cell polarity in mouse metaphase II oocytes: involvement of microtubules, microfilaments, and chromosomes.

A previous study showed that with hypertonic sucrose treatment, a projection is formed in mouse metaphase II (MII) oocytes in proximity to the spindle and chromosomes, where a polarized cortical domain is located. However, little is known about the mechanisms involved in this process. Here, we designed a series of experiments to test the hypothesis that hypertonicity is the induction factor for the formation of projections in mouse MII oocytes. Our hypothesis was supported by the following evidence: 1) different concentrations of sucrose affected the formation and shape of projections, whereas serum or basic media had little effect; 2) other hypertonic sugar solutions could also induce projection formation; and 3) projections could also be induced by hypertonic NaCl solution. We then tested the hypothesis that the cytoskeleton was involved in the formation of hypertonicity-induced projections. This was investigated by culturing MII- and germinal vesicle-stage mouse oocytes in the presence or absence of cytoskeletal inhibitors, including cytochalasin B (disruption of actin filaments), nocodazole (disruption of microtubules), and taxol (polymerization of tubulin molecules). We found that none of the cytoskeletal inhibitors alone could prevent hypertonicity-induced projection formation, whereas the combination of cytochalasin B with nocodazole or with taxol blocked the formation of these projections in most matured oocytes. When immature oocytes were incubated in cytochalasin B, but not in nocodazole or taxol, the formation of an actin-rich domain and the peripheral positioning of the spindle were blocked during maturation; hence, no projections were formed, even after hypertonic sucrose treatment. Based on these observations, we propose that three components are necessary for projection formation: 1) a polarized cortical patch (e.g., an actin-rich domain), 2) rigid submembrane structures (e.g., a spindle and/or chromosomes), and 3) solid connections between the above. Any disturbance of one of these factors will affect the hypertonicity-induced projection formation. Hypertonicity-induced projection in mouse oocytes thus provides an experimental model for studies regarding cell polarity and the interaction between membrane and submembrane components.

Role of facilitative glucose transporters in diffusional water permeability through J774 cells.

We have reported previously that in the presence of an osmotic gradient, facilitative glucose transporters (GLUTs) act as a transmembrane pathway for water flow. Here, we find evidence that they also allow water passage in the absence of an osmotic gradient. We applied the linear diffusion technique to measure the diffusional permeability (Pd) of tritiated water (3H-H2O) through plasma membranes of J774 murine macrophage-like cells. Untreated cells had a Pd of 30.9 +/- 1.8 microns/s; the inhibitors of facilitative glucose transport cytochalasin B (10 microM) and phloretin (20 microM) reduced that value to 15.3 +/- 1.8 (50%) and 11.0 +/- 0.7 (62%) microns/s, respectively. In contrast, no significant effect on Pd was observed in cells treated with dihydrocytochalasin B (Pd = 28.4 +/- 1.5 microns/s). PCMBS (3 mM) inhibited glucose uptake by greater than 95%, and 3H-H2O diffusion by approximately 30% (Pd = 22.9 +/- 1.5 microns/s). The combination of cytochalasin B plus pCMBS reduced Pd by about 87% (Pd = 3.9 +/- 0.3 microns/s). Moreover, 1 mM pCMBS did not affect the osmotic water permeability in Xenopus laevis oocytes expressing the brain/erythroid form of facilitative glucose transporters (GLUT1). These results indicate for the first time that about half of the total Pd of J774 cells may be accounted for by water passage across GLUTs. Hence, they highlight the multifunctional properties of these transporters serving as conduits for both water and glucose. Our results also suggest for the first time that pCMBS blocks glucose transport without affecting water permeation through GLUTs. Lastly, because pCMBS decreases the Pd of J774 cells, this suggests the presence in their plasma membranes of another protein(s) exhibiting water channel properties.

Disorganization of microfilaments and intermediate filaments interferes with the assembly and stability of desmosomes in MDCK epithelial cells

To investigate the possible role(s) of cytoskeletal elements in desmosome assembly we have studied the effects of cytostatic drugs on the assembly of desmosomes in MDCK epithelial cells. We showed previously [Pasdar et al.: Cell Motil. Cytoskeleton 23:201-213, 1992] that selective disruption of microtubules has no effect on desmosome assembly. Here, we have treated MDCK cells with cytochalasin B and a combination of cytochalasin B and nocodazole and analysed the effects of desmosome assembly. Immunofluorescence analysis of MDCK cultures following drug treatment indicated complete disruption of actin microfilaments and disorganization of cytokeratin intermediate filaments. Biochemical analysis of newly synthesized desmosomal membrane core glycoproteins as well as the cell adhesion protein E-cadherin revealed no effect of these drugs on the kinetics of synthesis, intracellular processing, or transport to the plasma membrane either in the presence or absence of cell-cell contact. However, morphological analyses revealed a significant disruption in the spatial organization of desmosomal proteins and E-cadherin. Drug treatment in the absence of cell-cell contact resulted in the disruption of the normally observed homogeneous punctate staining pattern and appearance of aggregate staining. Induction of cell-cell contact in these cultures resulted in redistribution of some of the aggregate staining to the plasma membrane. In contrast to control cultures, significant amount of intracellular staining was retained for all desmosomal proteins. Biochemical analyses of turnover rates of newly synthesized desmosomal proteins indicated a significant decrease in metabolic stability of these proteins while the turnover rate of E-cadherin was not significantly different among control and drug-treated cultures. Taken together, these results suggest that intact actin and cytokeratin filaments are necessary for the stability, efficient assembly, and spatial organization of the junctional components at the membrane. The regulatory role of cytokeratins and actin filaments in assembly and stability of desmosomes on the plasma membrane is discussed.