phalloidine and Osteosarcoma

Osteosarcoma (OS) is a highly metastatic bone cancer that usually affects children. Rhizoma Paridis saponins (RPS) have been identified to show a broad-spectrum anti-tumor activity. Our previous study has identified vasculogenic mimicry (VM) as an indicator of poor prognosis for OS. Rhizoma Paridis ethanol extract exhibits potent anti-OS property. However, the anti-metastatic effect of RPS on OS and the detailed mechanisms remain unknown. RPS was characterized by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS) analysis. The anti-OS, anti-metastasis and anti-VM activities of RPS were investigated using in vitro biological assays and a xenograft mouse model. Western blot, qRT-PCR, ELISA, Phalloidin staining and immunohistochemistry assays were conducted to investigate the molecular mechanism of RPS. A total of 34 phytochemicals from RPS were identified by LC/Q-TOF/MS. RPS dose-dependently suppressed the OS cell proliferation, metastasis and VM formation in vitro and in vivo. Mechanically, we found that RPS downregulated migration-inducing gene 7 (MIG-7) expression, resulting in inhibition of the PI3K/MMPs/Ln-5γ2 pathway and cell protrusion formation. Additionally, we confirmed that RPS downregulated MIG-7 by upregulating miR-520d-3p expression. Our results suggests that RPS inhibits the VM formation and metastasis of OS by modulating the miR-520d-3p/MIG-7 signaling axis.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Ethanol; Humans; Mice; MicroRNAs; Osteosarcoma; Phalloidine; Phosphatidylinositol 3-Kinases; Plant Extracts; Saponins

This paper examines the effects of nano-scale titanium coatings, and micro-groove/micro-grid patterns on cell/surface interactions on silicon surfaces. The nature of the cellular attachment and adhesion to the coated/uncoated micro-textured surfaces was elucidated by the visualization of the cells and relevant cytoskeletal & focal adhesion proteins through scanning electron microscopy and immunofluorescence staining. Increased cell spreading and proliferation rates are observed on surfaces with 50 nm thick Ti coatings. The micro-groove geometries have been shown to promote contact guidance, which leads to reduced scar tissue formation. In contrast, smooth surfaces result in random cell orientations and the increased possibility of scar tissue formation. Immunofluorescence cell staining experiments also reveal that the actin stress fibers are aligned along the groove dimensions, with discrete focal adhesions occurring along the ridges, within the grooves and at the ends of the cell extensions. The implications of the observed cell/surface interactions are discussed for possible applications of silicon in implantable biomedical systems.

Topics: Actins; Cell Adhesion; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Cell Size; Coated Materials, Biocompatible; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Imaging, Three-Dimensional; Lasers; Microscopy, Fluorescence; Models, Biological; Nanotechnology; Osteoblasts; Osteosarcoma; Phalloidine; Rhodamines; Silicon; Surface Properties; Titanium; Vinculin

CKIP-1 is a pleckstrin homology domain-containing protein that induces alterations of the actin cytoskeleton and cell morphology when expressed in human osteosarcoma cells. CKIP-1 interacts with the heterodimeric actin-capping protein in cells, so we postulated that this interaction was responsible for the observed cytoskeletal and morphological effects of CKIP-1. To test this postulate, we used peptide "walking arrays" and alignments of CKIP-1 with CARMIL, another CP-binding protein, to identify Arg-155 and Arg-157 of CKIP-1 as residues potentially required for its interactions with CP. CKIP-1 mutants harboring Arg-155 and Arg-157 substitutions exhibited greatly decreased CP binding, while retaining wild-type localization, the ability to interact with protein kinase CK2, and self-association. To examine the phenotype associated with expression of these mutants, we generated tetracycline-inducible human osteosarcoma cells lines expressing R155E,R157E mutants of CKIP-1. Examination of these cell lines reveals that CKIP-1 R155E,R157E did not induce the distinct changes in cell morphology and the actin cytoskeleton that are characteristic of wild-type CKIP-1 demonstrating that the interaction between CKIP-1 and CP is required for these cellular effects.

Topics: Actins; Amino Acid Sequence; Animals; Arginine; Carrier Proteins; Casein Kinase II; Cell Line, Tumor; Humans; Intracellular Signaling Peptides and Proteins; Molecular Sequence Data; Mutation; Osteosarcoma; Peptides; Phalloidine; Protein Binding; Sequence Homology, Amino Acid

Effects of T8993G mutation in mitochondrial DNA (mtDNA), associated with neurogenical muscle weakness, ataxia and retinitis pigmentosa (NARP), on the cytoskeleton, mitochondrial network and calcium homeostasis in human osteosarcoma cells were investigated. In 98% NARP and rho(0) (lacking mtDNA) cells, the organization of the mitochondrial network and actin cytoskeleton was disturbed. Capacitative calcium entry (CCE) was practically independent of mitochondrial energy status in osteosarcoma cell lines. The significantly slower Ca(2+) influx rates observed in 98% NARP and rho(0), in comparison to parental cells, indicates that proper actin cytoskeletal organization is important for CCE in these cells.

Topics: Actins; Ataxia; Benzimidazoles; Calcium; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line, Tumor; Cytoskeleton; DNA, Mitochondrial; Fluorescent Dyes; Fura-2; Humans; Image Processing, Computer-Assisted; Immunohistochemistry; Ionophores; Membrane Potentials; Microscopy, Fluorescence; Mitochondrial Myopathies; Muscle Weakness; Mutation; Osteosarcoma; Phalloidine; Retinitis Pigmentosa; Rhodamines; Thapsigargin

An in vitro mineralizing cell-implant system was developed to study osteoblast attachment, secretion of extracellular (ECM) matrix proteins and mineralization. Saos-2 cells were plated on Tivanium (Tiv, Ti-6A1-4V), Zimaloy (Zim, Co-Cr-Mo) and glass disks. The cells were cultured in alpha-MEM medium with 10% fetal bovine serum and 50 microg ml(-1) ascorbic acid. The cultures were analyzed for calcification and for mRNA expression for ECM proteins after 1, 2, 4 and 6 weeks. Calcium content was significantly higher in cells on Tiv, less on Zim and least on glass disks. With the addition of 3 mm beta-glycerophosphate (beta-GP), the cell layer was more calcified on Zim than on Tiv and all substrates had three times more calcium than cultures without beta-GP. All subsequent experiments were performed without beta-GP. Phalloidin immunofluorescence microscopy of the actin-based cytoskeleton at 2 weeks demonstrated nodules composed of multilayered, cobblestone-appearing osteoblasts overlying calcified matrix which was stained with calcein. On Tiv, calcified nodules were connected in a trabecular-like pattern while on Zim, calcification was dispersed throughout the cell layer. Northern blots for alkaline phosphatase, bone sialoprotein, osteocalcin and alpha1(I) procollagen mRNAs were performed at different time points. The amount and pattern of calcification as well as the expression of ECM-mRNAs differed on each implant material. The results indicate that Tiv stimulates the production of more ECM proteins and mineralized matrix than Zim or glass in this osteoblast-like cell/implant culture.

Topics: Alloys; Biocompatible Materials; Blotting, Northern; Calcification, Physiologic; Calcium; Cell Adhesion; Chromium Alloys; Fluoresceins; Fluorescent Dyes; Humans; Osseointegration; Osteoblasts; Osteosarcoma; Phalloidine; Prostheses and Implants; Titanium; Tumor Cells, Cultured