cytochalasin-b and Osteosarcoma

Mammalian cells take in D-glucose as an essential fuel as well as a carbon source. In contrast, L-glucose, the mirror image isomer of D-glucose, has been considered merely as a non-transportable/non-metabolizable control for D-glucose. We have shown that 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), a D-glucose analogue combining a fluorophore NBD at the C-2 position, is useful as a tracer for monitoring D-glucose uptake through glucose transporters (GLUTs) into mammalian cells. To more precisely evaluate the stereoselectivity of 2-NBDG uptake, we developed an L-glucose analogue 2-NBDLG, the mirror-image isomer of 2-NBDG. Interestingly, 2-NBDLG was taken up into mouse insulinoma MIN6 cells showing nuclear heterogeneity, a cytological feature of malignancy, while remaining MIN6 cells only exhibited a trace amount of 2-NBDLG uptake. The 2-NBDLG uptake into MIN6 cells was abolished by phloretin, but persisted under blockade of major mammalian glucose transporters. Unfortunately, however, no such uptake could be detected in other tumor cell lines. Here we demonstrate that human osteosarcoma U2OS cells take in 2-NBDLG in a phloretin-inhibitable manner. The uptake of 2-NBDG, and not that of 2-NBDLG, into U2OS cells was significantly inhibited by cytochalasin B, a potent GLUT inhibitor. Phloretin, but neither phlorizin, an inhibitor of sodium-glucose cotransporter (SGLT), nor a large amount of D/L-glucose, blocked the 2-NBDLG uptake. These results suggest that a phloretin-inhibitable, non-GLUT/non-SGLT, possibly non-transporter-mediated yet unidentified mechanism participates in the uptake of the fluorescent L-glucose analogue in two very different tumor cells, the mouse insulinoma and the human osteosarcoma cells.

Topics: 4-Chloro-7-nitrobenzofurazan; Animals; Bone Neoplasms; Cytochalasin B; Deoxyglucose; Depression, Chemical; Glucose; Glucose Transport Proteins, Facilitative; Humans; Insulinoma; Isomerism; Mice; Osteosarcoma; Pancreatic Neoplasms; Phloretin; Sodium-Glucose Transporter 2 Inhibitors; Tumor Cells, Cultured

Subcellular organelle dynamics are strongly influenced by interactions with cytoskeletal filaments and their associated motor proteins, and lead to complex multiexponential relaxations that occur over a wide range of spatial and temporal scales. Here we report spatio-temporal measurements of the fluctuations of the mitochondrial reticulum in osteosarcoma cells by using Fourier imaging correlation spectroscopy, over time and distance scales of 10(-2) to 10(3) s and 0.5-2.5 microm. We show that the method allows a more complete description of mitochondrial dynamics, through the time- and length-scale-dependent collective diffusion coefficient D(k,tau), than available by other means. Addition of either nocodazole to disrupt microtubules or cytochalasin D to disassemble microfilaments simplifies the intermediate scattering function. When both drugs are used, the reticulum morphology of mitochondria is retained even though the cytoskeletal elements have been de-polymerized. The dynamics of the organelle are then primarily diffusive and can be modeled as a collection of friction points interconnected by elastic springs. This study quantitatively characterizes organelle dynamics in terms of collective cytoskeletal interactions in living cells.

Topics: Actin Cytoskeleton; Bone Neoplasms; Cytochalasin B; Cytoskeleton; Fourier Analysis; Humans; Microtubules; Mitochondria; Nocodazole; Osteosarcoma; Tumor Cells, Cultured

Thyroid hormone deficient osteoblastic cells in cell culture released a significantly higher amount of alkaline phosphate (ALP) activity following T3 replacement. T3 increased the release of total and membrane-bound ALP activity in these cells significantly more than T4 or inactive thyroid hormone metabolite, DIT. The effect of T3 on the membrane-bound ALP fraction was dose and time dependent; higher concentrations of T3 and longer incubation time with T3 proportionally increased the enzyme activity. T3 had no effect on the release of soluble fraction of ALP. Our results indicate that in "hypothyroid" osteoblastic cells the total release of ALP is decreased and that the secreted fraction of ALP is predominantly in soluble form, whereas the addition of T3 stimulates ALP release and mainly increases the membrane-bound fraction. T3 also increased formation of actin cytoskeleton in hypothyroid osteoblastic cells. Cytochalasin treatment, through its inhibition of actin polymerization, produced a significant decrease of membrane-bound ALP release induced by T3. These data suggest that the regulatory role of T3 in skeletal development can partly be due to its stimulatory effect on the release of membrane-bound ALP by osteoblastic cells which is thought to be an important factor in the initiation of biological calcification.

Topics: Actins; Alkaline Phosphatase; Animals; Cell Count; Cell Fractionation; Cell Membrane; Cytochalasin B; Cytoskeleton; Diiodothyronines; Dose-Response Relationship, Drug; Osteoblasts; Osteosarcoma; Rats; Thyroxine; Time Factors; Triiodothyronine; Tumor Cells, Cultured

We have previously reported that P-glycoprotein (Pgp)-overexpressing multidrug resistant (MDR) osteosarcoma cells were functionally more differentiated than their parent cells. The present study showed that in the parent cells, the actin filaments were sparsely distributed or were diffusely spread throughout the cytoplasm, whereas the MDR osteosarcoma cells exhibited a remarkable increase in well-organized actin stress fibers. Furthermore, dihydrocytochalasin B, a specific inhibitor of actin polymerization, dramatically disrupted this network of stress fibers, increased the intracellular accumulation of doxorubicin (DOX) and modified the resistance against DOX. These results indicate that the organization of actin filaments associated with cellular differentiation may be involved in the expression of Pgp function in the MDR osteosarcoma cells.

Topics: Actins; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Differentiation; Cytochalasin B; Doxorubicin; Drug Resistance, Multiple; Mice; Osteosarcoma; Tumor Cells, Cultured