Nanoparticles Containing Curcumin Useful for Suppressing Macrophages In Vivo in Mice.

To explore a novel method using liposomes to suppress macrophages, we screened food constituents through cell culture assays. Curcumin was one of the strongest compounds exhibiting suppressive effects on macrophages. We subsequently tried various methods to prepare liposomal curcumin, and eventually succeeded in preparing liposomes with sufficient amounts of curcumin to suppress macrophages by incorporating a complex of curcumin and bovine serum albumin. The diameter of the resultant nanoparticles, the liposomes containing curcumin, ranged from 60 to 100 nm. Flow cytometric analyses revealed that after intraperitoneal administration of the liposomes containing curcumin into mice, these were incorporated mainly by macrophages positive for F4/80, CD36, and CD11b antigens. Peritoneal cells prepared from mice injected in vivo with the liposomes containing curcumin apparently decreased interleukin-6-producing activities. Major changes in body weight and survival rates in the mice were not observed after administrating the liposomes containing curcumin. These results indicate that the liposomes containing curcumin are safe and useful for the selective suppression of macrophages in vivo in mice.

Evaluation of selective tumor detection by clinical magnetic resonance imaging using antibody-conjugated superparamagnetic iron oxide.

Active targeting by monoclonal antibodies (mAbs) combined with nanosize superparamagnetic iron oxide (SPIO) is a promising technology for magnetic resonance imaging (MRI) diagnosis. However, the clinical applicability of this technology has not been investigated using appropriate controls. It is important to evaluate the targeting technology using widely used clinical 1.5-Tesla MRI in addition to the high-Tesla experimental MRI. In this study, we measured mAb-conjugated dextran-coated SPIO nanoparticles (CMDM) in vivo using clinical 1.5-Tesla MRI. MRI of tumor-bearing mice was performed using a simple comparison between positive and negative tumors derived from the same genetic background in each mouse. The system provided significant tumor-targeting specificity of the target tumor. To the best of our knowledge, this is the first report on the specific detection of target tumors by mAb-conjugated SPIO using clinical 1.5-Tesla MRI. Our observations provide clues for reliable active targeting using mAb-conjugated SPIO in clinical applications.

E-selectin targeting to visualize tumors in vivo.

Generally angiogenic factors induce the expression of E-selectin in vascular endothelial cells in the tumors. In this study, we employed an anti-E-selectin monoclonal antibody to target tumors in vivo and evaluated an optical imaging reagent to visualize tumor regions. The anti-E-selectin antibody was conjugated on the surface of liposomes, which encapsulated the near-infrared fluorescent substances Cy3 or Cy5.5. The liposomes efficiently recognized human umbilical vein endothelial cells only when E-selectin was induced by angiogenic factors such as TNF-alpha in vitro. Cy5.5 encapsulated into liposomes that were conjugated with an anti-E-selectin antibody successfully visualized Ehrlich ascites tumor cells when transplanted into mice. Thus, E-selectin targeting with liposomes containing a near-infrared fluorescent dye was found effective in visualizing tumors in vivo. This strategy should be extremely useful as a method to identify sentinel lymphatic nodes and angiogenic tumors as well as use for drug delivery to tumor cells.

Role of nm23 in the regulation of cell shape and migration via Rho family GTPase signals.

Rho family small GTPase plays a key role in the regulation of cell shape and migration in mammalian cells. Constitutive activation of Rho GTPase leads to the aberrant cell morphology and migration. We identified nm23-H2 as a binding partner of Lbc proto-oncogene product, which specifically activates RhoA, and revealed that nm23-H2 could act as a negative regulator of Rho activity. Furthermore, we found that Lbc, nm23-H2 and ICAP1-alpha could form tertial complex in cells, and this complex formation was thought to be critical for cell migration stimulated by integrin. It is reported that nm23-H1 bound to Tiam1 and Dbl, which activates Rac and Cdc42 small GTPase, respectively. We discuss the role of nm23 in the regulation of cell morphology and cell migration via Rho family GTPases.

Lbc proto-oncogene product binds to and could be negatively regulated by metastasis suppressor nm23-H2.

Lbc was identified as transforming gene from human leukemic cells and encodes Rho type guanine nucleotide exchange factor with 47kDa molecular weight. We isolated overlapping cDNAs of Lbc from human lung tissue. Full-length Lbc cDNA encodes 309kDa huge protein with Ht31 PKA anchoring motif, Dof domain, C1 domain, and coiled-coil structure. In order to analyze the regulatory mechanism of its activity, we searched for binding proteins. By yeast two-hybrid screening, we identified metastasis suppressor nm23-H2 as binding protein, which interacts with amino-terminal region of Lbc containing Dof domain. nm23 gene family encodes nucleoside diphosphate kinase, however, the binding of nm23-H2 to Lbc was independent of kinase activity. nm23-H1, which binds to Rac-specific GEF Tiam1, could not bind to Lbc suggesting nm23-H2 would be specific regulator for Lbc. Expression of nm23-H2 in cells leads to decrease the amount of GTP-bound Rho and suppress stress fiber formation stimulated by expression of Lbc. Our data suggest that metastasis suppressor nm23-H2 could regulate Lbc negatively by binding to amino-terminal region of Lbc proto-oncogene product.