carbocyanines and Osteosarcoma

Osteosarcoma (OS) is one of most malignant bone tumors in early adolescence, which is a highly metastatic cancer and pulmonary metastasis is the most common cause of death. Thus, the development of efficient approaches to discover potential compounds that target metastasis of OS remains a topic of considerable interest. In this study, subtractive Cell-SELEX was performed to screen OS metastasis specific DNA aptamers by using cell lines with similar tumorigenic potentials but opposite metastatic aggressiveness (highly metastatic 143B cells and non-metastatic U-2 OS cells as the target and negative cells, respectively). This in vitro selection generated an ssDNA aptamer LP-16 that exhibited high binding affinity to 143B cells with an equilibrium dissociation constant (K

Topics: Animals; Aptamers, Nucleotide; Carbocyanines; Cell Line, Tumor; DNA, Single-Stranded; Female; Flow Cytometry; Fluorescent Dyes; Humans; Mice, Inbred BALB C; Microscopy, Confocal; Microscopy, Fluorescence; Molecular Probes; Neoplasm Metastasis; Osteosarcoma; SELEX Aptamer Technique

About 40% of osteosarcoma patients die of metastases. Novel strategies to improve treatment of metastatic patients require a better understanding of the processes involved, like angiogenesis, migration, and the immune response. However, the rarity of osteosarcoma and its heterogeneity make this neoplasm difficult to study. Recently we reported malignant transformation of mouse mesenchymal stem cells (MSCs) which formed osteosarcoma upon transplantation into mice. Here we studied these cells in zebrafish embryos and found that transformed MSCs induced angiogenesis and migrated through the bodies of the embryos, but this was never observed with non-transformed normal MSCs (progenitors of the transformed MSCs). Whole genome expression analysis of both the cells and the host showed that angiogenesis and migration-related genes matrix metalloproteinase 19 (Mmp-19) and erythroblastosis virus E26 oncogene homologue 1 (Ets-1) were overexpressed in transformed MSCs compared to normal MSCs. Investigating the host response, embryos injected with transformed MSCs showed decreased expression of immune response-related genes, especially major histocompatibility complex class 1 (mhc1ze), as compared to embryos injected with normal MSCs. These findings contribute to the identification of genetic events involved in angiogenesis, migration, and host response providing targets as well as an appropriate model for high-throughput drug screens.

Topics: Animals; Animals, Genetically Modified; Bone Neoplasms; Carbocyanines; Cell Movement; Cell Transformation, Neoplastic; Cells, Cultured; Fluorescent Dyes; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Luminescent Proteins; Matrix Metalloproteinases, Secreted; Mesenchymal Stem Cell Transplantation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Invasiveness; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Osteosarcoma; Proto-Oncogene Protein c-ets-1; Red Fluorescent Protein; Time Factors; Tumor Escape; Zebrafish; Zebrafish Proteins

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

Severe disruption of mitochondrial function is generally considered to provide a powerful trigger for apoptosis in mammalian cells. We report here that intact cells may undergo the mitochondrial permeability transition and mitochondria swell in a fully reversible manner, without inducing cell death. Cultured human osteosarcoma cells (143B TK-) stained with JC-1, MitoTracker dyes, or calcein plus Co2+ were imaged by confocal microscopy to visualize changes of mitochondrial membrane potential (DeltaPsim), morphology, and permeability transition, respectively, during treatment with a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells rapidly exhibited mitochondrial permeability transition and swelling after addition of CCCP, but the swelling subsided within hours, leaving mitochondria that appeared in punctate form, not filamentous as before CCCP treatment. Cyclosporin A impeded the permeability transition and swelling, although complete inhibition was not observed. Cells survived the dissipation of DeltaPsim by CCCP for up to 6 h without developing any obvious cell damage or signs of apoptosis. With the restoration of DeltaPsim after removal of CCCP (following 6 h of CCCP treatment), permeability transition pores were closed. These results suggest that none of the following events represent a point of no return in the process of apoptotic cell death: loss of DeltaPsim, mitochondrial permeability transition, or mitochondrial swelling.

Topics: Aldehydes; Apoptosis; Benzimidazoles; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Survival; Cyclosporine; Fluoresceins; Fluorescent Dyes; Humans; Intracellular Membranes; Ionophores; Membrane Potentials; Microscopy, Confocal; Mitochondria; Mitochondrial Swelling; Organic Chemicals; Osteosarcoma; Permeability; Staining and Labeling; Tumor Cells, Cultured