phalloidine and thiazolyl-blue

Simvastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, and widely used as cholesterol-lowering agent, has been suggested for its beneficial effects on alveolar bone formation, regeneration of dental pulp tissue and periodontal ligament. High doses of simvastatin appear to induce apoptosis in several cell types, but little is known about its possible effect on tooth-associated cells. Therefore, the effects of simvastatin were studied on apoptosis and cell morphology of human dental pulp cells (HDPCs) and periodontal ligament fibroblasts (HPLFs).. HDPCs/HPLFs obtained from 4 patients were cultured with or without various concentrations of simvastatin (0.1, 1, and 10μM) for 24, 48, and 72h. The 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate cell viability. The levels of apoptosis of HDPCs and HPLFs were measured by flow cytometry after Annexin V/propidium iodide double staining. Phalloidin-FITC and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) staining was used to examine differences in the actin cytoskeleton and nuclear morphology, respectively.. The viability of HDPCs and HPLFs was significantly reduced after simvastatin treatment in a dose- and time-dependent manner (p<0.05). The apoptosis of HDPCs and HPLFs was significantly increased in 10μM simvastatin-treated cells (p<0.05). The effect on apoptosis was comparable for HDPCs and HPLFs. Nuclear staining showed typical apoptotic nuclear condensation and fragmentation in simvastatin-treated HDPCs/HPLFs. A dose- and time-dependent simvastatin-induced disruption of the actin cytoskeleton was observed in both cell types.. Our data demonstrated that simvastatin decreases the viability of HDPCs and HPLFs, probably by inducing apoptosis.

Topics: Actin Cytoskeleton; Adolescent; Adult; Apoptosis; Cell Culture Techniques; Cell Nucleus; Cell Shape; Cell Survival; Cells, Cultured; Coloring Agents; Dental Pulp; Dose-Response Relationship, Drug; Fibroblasts; Flow Cytometry; Fluorescent Dyes; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; Periodontal Ligament; Phalloidine; Simvastatin; Tetrazolium Salts; Thiazoles; Time Factors; Young Adult

Mineral fibers are potential carcinogens to humans. In order to help clarify the etiology of the pathological effects of asbestos, cellular reactions to natural and synthetic asbestos fibers were compared using a lung alveolar cancer cell line (A549 epithelial cells), considered the first target of inhaled micro-environmental contaminants. Natural asbestos tremolite (NAT) fibers were collected from rocks in NW Italy. Synthetic asbestos tremolite (SAT) was iron-free and therefore considered as standard tremolite. Both fibers, subjected to mineralogical characterization by X-ray powder diffractometry, electron microscopy and energy dispersive spectrometry, fell within the definition of respirable and potentially carcinogenic fibers. Several signs of functional and structural cell damage were found after treatment with both fibers, documented by viability, motility, and morphological perturbations. Phalloidin labeling showed irregular distribution of cytoskeletal F-actin, whereas immunohistochemical investigations showed abnormal expression of VEGF, Cdc42, β-catenin, assessed as risks indicators for cancer development. Both fibers caused significant loss of viability, even compared to UICC crocidolite, but, while SAT fibers exerted a more direct cytotoxic effect, survival of damaged cells expressing high VEGF levels was detected after NAT contact. This in vitro pilot study outlines potential health risks of NAT fibers in vivo related to their iron content, which could trigger signaling networks connected with cell proliferation and neoplastic transformation.

Topics: Actins; Apoptosis; Asbestos; Asbestos, Amphibole; Asbestos, Crocidolite; beta Catenin; cdc42 GTP-Binding Protein; Cell Line, Tumor; Cell Survival; Cytoskeleton; Humans; Immunohistochemistry; Iron; Microscopy, Electron; Mitosis; Necrosis; Phalloidine; Pilot Projects; Tetrazolium Salts; Thiazoles; Time Factors; Vascular Endothelial Growth Factor A; X-Rays

The apomyoglobin mutant W7FW14F forms amyloid-like fibrils at physiological pH. We examined the kinetics of fibrillogenesis using three techniques: the time dependence of the fluorescence emission of thioflavin T and 1-anilino-8-naphthalenesulfonate, circular dichroism measurements, and electron microscopy. We found that in the early stage of fibril formation, non-native apomyoglobin molecules containing beta-structure elements aggregate to form a nucleus. Subsequently, more molecules aggregate around the nucleus, thereby resulting in fibril elongation. We evaluated by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) the cytotoxicity of these aggregates at the early stage of fibril elongation versus mature fibrils and the wild-type protein. Similar to other amyloid-forming proteins, cell toxicity was not due to insoluble mature fibrils but rather to early pre-fibrillar aggregates. Propidium iodide uptake showed that cell toxicity is the result of altered membrane permeability. Phalloidin staining showed that membrane damage is not associated to an altered cell shape caused by changes in the cytoskeleton.

Topics: Anilino Naphthalenesulfonates; Animals; Apoproteins; Benzothiazoles; Cell Membrane; Cell Nucleus; Circular Dichroism; Coloring Agents; Cytoskeleton; Fluorescent Dyes; Hydrogen-Ion Concentration; Kinetics; Mice; Microscopy, Electron; Mutation; Myoglobin; NIH 3T3 Cells; Phalloidine; Propidium; Protein Structure, Secondary; Spectrometry, Fluorescence; Tetrazolium Salts; Thiazoles; Time Factors; Ultraviolet Rays