cytochalasin-d and Osteosarcoma

Microfluidics, the science of engineering fluid streams at the micrometer scale, offers unique tools for creating and controlling gradients of soluble compounds. Gradient generation can be used to recreate complex physiological microenvironments, but is also useful for screening purposes. For example, in a single experiment, adherent cells can be exposed to a range of concentrations of the compound of interest, enabling high-content analysis of cell behaviour and enhancing throughput. In this study, we present the development of a microfluidic screening platform where, by means of diffusion, gradients of soluble compounds can be generated and sustained. This platform enables the culture of adherent cells under shear stress-free conditions, and their exposure to a soluble compound in a concentration gradient-wise manner. The platform consists of five serial cell culture chambers, all coupled to two lateral fluid supply channels that are used for gradient generation through a source-sink mechanism. Furthermore, an additional inlet and outlet are used for cell seeding inside the chambers. Finite element modeling was used for the optimization of the design of the platform and for validation of the dynamics of gradient generation. Then, as a proof-of-concept, human osteosarcoma MG-63 cells were cultured inside the platform and exposed to a gradient of Cytochalasin D, an actin polymerization inhibitor. This set-up allowed us to analyze cell morphological changes over time, including cell area and eccentricity measurements, as a function of Cytochalasin D concentration by using fluorescence image-based cytometry.

Topics: Cell Culture Techniques; Cell Line, Tumor; Cytochalasin D; Humans; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques; Optical Imaging; Osteosarcoma; Shear Strength

Cell mechanical properties that depend on cytoskeleton architecture are critical to the mechanotransduction process, and have great potential for cancer diagnosis and therapy. In this study, the morphological and mechanical properties of typical osteosarcoma microenvironment cells, including mesenchymal stem cells (MSC), normal human osteoblast cells (NHOst) and osteosarcoma cells (MG-63), were compared using atomic force microscopy (AFM). The MG-63 cells were smaller and thicker than the MSC and NHOst cells. The membrane roughness of MG-63 cells was higher than that of MSC and NHOst cells. The MG-63 cells had lower stiffness than their normal counterparts due to their reduced organization of the cytoskeleton structure. The cell stiffness influenced the mechanotransduction. The MG-63 cells had a lower percentage of nuclear YAP/TAZ compared with the MSC and NHOst cells. The F-actin assembly was disrupted by the cytochalasin D (cyto D) treatment used to investigate its influence on mechanotransduction. Disruption of the cytoskeleton leaded to a decrease of the cell stiffness, and reduced the nuclear YAP/TAZ percentage, indicating its inhibition in the cell mechanotransduction process. This study would shed light on the development of a novel cancer diagnosis strategy and would contribute to reveal the relationship between the cytoskeleton structure and the cell mechanical properties.

Topics: Actins; Bone Neoplasms; Cell Line, Tumor; Cells, Cultured; Cytochalasin D; Cytoskeleton; Humans; Mechanotransduction, Cellular; Mesenchymal Stem Cells; Microscopy, Atomic Force; Osteoblasts; Osteosarcoma; Tumor Microenvironment

As the outmost coating of cells, the pericellular matrix (PCM) involved in various cellular functions has been exploited previously to be able to accumulate 120 nm Au nanoparticles (NPs), adjust their diffusion coefficient similar to that of membrane receptors, and enhance their uptake efficiency. In this study, the interactions between PCM and NPs with different sizes and materials were systematically investigated. We found that PCM can selectively enhance the retention and cellular uptake of NPs with diameters from 50 to 180 nm, but has no enhancement effect for 20 nm NPs. Identical behaviors of PCM was observed for both Au NPs and polystyrene NPs, indicating that this unique phenomenon is more related to the dimensions of the NPs. The study of single-particle tracking of 50-180 nm NPs on the surface of thick PCM cells revealed that PCM actively adjusts the diffusion coefficient of NPs to ∼0.1 μm(2)/s regardless of their sizes. By blocking the receptor-mediated endocytosis (RME) pathway with four different inhibitors, this active role of PCM can be effectively suppressed, further confirming that the trapping and retention of NPs by PCM is an inherent biological function. These findings provided new insights for better understanding of the RME pathway and may have promising NP-based applications for controlled drug delivery and therapy in biomedicine.

Topics: Bone Neoplasms; Cytochalasin D; Drug Carriers; Endocytosis; Gold; HeLa Cells; Humans; Metal Nanoparticles; Nucleic Acid Synthesis Inhibitors; Osteosarcoma; Particle Size; Surface Properties; Tumor Cells, Cultured

The tumor suppressor p21WAF/CIP1 mediates the proliferation arrest via p53-dependent or -independent gene transactivation following distinct environmental stresses. In this study, we show that direct destabilization of the actin cytoskeleton by actin-targeting reagents leads to a p53-independent up-regulation of p21WAF/CIP1. The actin-targeting agent cytochalasin B (10 microM) quickly disrupted the actin cytoskeleton of p53 wild-type and p53-null cells accompanied by up-regulation of p21WAF/CIP1. Nevertheless, both total p53 and ser-15 phosphorylated p53 were not accumulated concomitantly, compared to the effect caused by ionizing irradiation. P53-independent up-regulation of p21WAF/CIP1 was also observed by two other actin-targeting agents cytochalasin D and latrunculin B, but not by the microtubule inhibitor colcemid. Furthermore, we showed that p21WAF/CIP1 mRNA level was not increased, whereas the protein degradation was delayed. A reduction of ubiquitination for p21WAF/CIP1 protein was detected using immunoprecipitation/immunoblot analysis. Up-regulation of p21WAF/CIP1 was not associated with cytotoxicity induced by cytochalasin B that influenced DNA integrity and plating efficiency only after 24 h of treatment. In addition, up-regulated p21WAF/CIP1 was accompanied by reduction of phosphorylation on retinoblastoma (Rb) protein in p53-null cells, implying that p21WAF/CIP1 might in part account for the molecular regulation of cytochalasin B induced G1 phase arrest. Together, current results suggest that p21WAF/CIP1 level can be mediated by actin organization in the absence of p53 via a post-transcriptional machinery, and it may contribute to the growth ablation by agents targeting the actin cytoskeleton.

Topics: Actins; Adenocarcinoma; Bone Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cytochalasin D; Cytoskeleton; DNA, Neoplasm; Genes, p53; Humans; Lung Neoplasms; Osteosarcoma; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA, Neoplasm; Thiazolidines; Ubiquitin

G-protein-coupled receptors can couple to different signal transduction pathways in different cell types (termed cell-specific signaling) and can activate different signaling pathways depending on the receptor conformation(s) stabilized by the activating ligand (functional selectivity). These concepts offer potential for developing pathway-specific drugs that increase efficacy and reduce side effects. Despite significant interest, functional selectivity has been difficult to exploit in drug discovery, in part due to the burden of multiple assays. Cellular impedance assays use an emerging technology that can qualitatively distinguish Gs, Gi/o, and Gq signaling in a single assay and is thereby suited for studying these pharmacological concepts. Cellular impedance confirmed cell-specific Gs and Gq coupling for the melanocortin-4 receptor and dual Gi and Gs signaling with the cannabinoid-1 (CB1) receptor. The balance of Gi versus Gs signaling depended on the cell line. In CB1-HEKs, Giand Gs-like responses combined to yield a novel impedance profile demonstrating the dynamic nature of these traces. Cellspecific signaling was observed with endogenous D1 receptor in U-2 cells and SK-N-MC cells, yet the pharmacological profile of partial and full agonists was similar in both cell lines. We conclude that the dynamic impedance profile encodes valuable relative signaling information and is sufficiently robust to help evaluate cell-specific signaling and functional selectivity.

Topics: alpha-MSH; Animals; Biological Assay; Brain Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Cells, Cultured; CHO Cells; Cricetinae; Cricetulus; Cytochalasin D; Dopamine Agonists; Dose-Response Relationship, Drug; Electric Impedance; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gq-G11; GTP-Binding Protein alpha Subunits, Gs; Humans; Inhibitory Concentration 50; Kidney; Neuroectodermal Tumors, Primitive, Peripheral; Osteosarcoma; Pertussis Toxin; Receptor, Muscarinic M1; Receptors, Dopamine D2; Receptors, Dopamine D5; Receptors, G-Protein-Coupled; Sensitivity and Specificity; Signal Transduction

Both transforming growth factor-beta (TGF-beta)-induced expression of biglycan (BGN) and activation of p38 MAPK have been implicated in cellular adhesion and migration. Here, we analyzed the role of adhesive events and the small GTPase Rac1 in TGF-beta regulation of BGN. TGF-beta1 induction of BGN expression and activation of p38 was abolished or strongly reduced when cells were kept in suspension or exposed to either the actin cytoskeleton-disrupting agent cytochalasin D or a specific chemical Rac1 inhibitor. Ectopic expression of a dominant negative mutant (T17N) of Rac1 abrogated both TGF-beta-induced p38 MAPK activation and BGN up-regulation but did not affect TGF-beta-induced phosphorylation of Smad3 or transcriptional induction of Growth Arrest DNA Damage 45beta, previously shown to be crucial for TGF-beta regulation of BGN. Overexpression of wild type Rac1 greatly enhanced the TGF-beta effect on BGN in adherent cells, whereas ectopic expression of constitutively active Rac1 (Q61L) activated p38 and in the presence of exogenous TGF-beta was able to rescue BGN expression in nonadherent cells. Endogenous Rac1 was activated by TGF-beta treatment in PANC-1 cells in an adhesion-dependent fashion. Like Rac1-T17N, the NADPH oxidase inhibitor diphenylene iodonium and the tyrosine kinase inhibitor herbimycin A blocked TGF-beta-induced p38 activation and BGN expression, suggesting that Rac1 exerts its effect on BGN and p38 through increasing NADPH oxidase activity and subsequent production of reactive oxygen species. These results show that the TGF-beta effect on BGN is dependent on cell adhesion and that activated Rac1, presumably acting through NADPH oxidase(s), is necessary but not sufficient for TGF-beta-induced BGN expression.

Topics: Biglycan; Carcinoma; Cell Adhesion; Cell Line, Tumor; Cytochalasin D; Enzyme Activation; Enzyme Inhibitors; Extracellular Matrix Proteins; Gene Expression Regulation; Humans; Models, Biological; Mutation; NADPH Oxidases; Nucleic Acid Synthesis Inhibitors; Onium Compounds; Osteosarcoma; Oxidation-Reduction; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Proteoglycans; rac1 GTP-Binding Protein; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

1,25-dihydroxyvitamin D3 produces pronounced shape changes in fetal rat calvaria and osteosarcoma-derived (ROS 17/2.8) osteoblastic cells, characterized by retracting processes and cell rounding followed by aggregation of cells. The 1,25(OH)2D3 effect on ROS 17/2.8 morphology was determined morphometrically on scanning electron micrographs. The hormone effect was found to be dose dependent between 10(-12) and 10(-9) M. The shape changes appeared 12 h after hormone (10(-10) M) addition and were present in 80% of the ROS 17/2.8 cells and in 50% of the calvaria cells at 72 h. Cycloheximide at 1 microM, inhibited the hormone-dependent change in morphology. The 1,25(OH)2D3 effects were partially mimicked by 10(-8) M 25(OH)D3 but not by 10(-10) M 25(OH)D3 or 10(-11)-10(-8) M 24,25(OH)2D3. 1,25-dihydroxyvitamin D3 also increased cell proliferation twofold at 14 days in serum-free medium. 1,25(OH)2D3 treatment produced changes in microfilament organization, visualized with rhodamine-conjugated phalloidin. Microfilaments were localized at the terminal attachment points and in the perinuclear region, and few if any, were seen in the retracting processes themselves. Estimation of cytoskeletal actin and myosin by gel electrophoresis of Triton X-100 nonextractable proteins showed a 30% reduction in these proteins in the hormone-treated cells. Microtubules visualized by indirect immunofluorescence showed no major changes in organization. Both colchicine and cytochalasin D altered the hormone-induced shape change, suggesting that both microfilaments and microtubules were required for this process. Thus, 1,25(OH)2D3 had pronounced effects on cell shape in osteoblastic cells, probably via de novo protein synthesis. These changes lead to rearrangement of the cytoskeleton, primarily the microfilaments.

Topics: Actins; Animals; Bone Neoplasms; Calcitriol; Cell Count; Cells, Cultured; Colchicine; Cycloheximide; Cytochalasin D; Cytochalasins; Cytoskeleton; Fluorescent Antibody Technique; Microscopy, Electron, Scanning; Microtubules; Myosins; Osteoblasts; Osteosarcoma; Rats; Rats, Inbred Strains; Skull; Tumor Cells, Cultured