Masato Kodaka

Significance of VLA-4–VCAM-1 interaction and CD44 for transendothelial invasion in a bone marrow metastatic myeloma model

In previous work, we established the B9/BM1 syngeneic murine bone marrow metastasis model. Interleukin (IL)-6-dependent, IL-1-producing B9/BM1 cells, which colonize the vertebral and femoral marrow after i.v. injection, show great similarity in cell surface phenotype to human myeloma cells, especially the expression of 3 adhesion molecules, CD44, VLA-4 and ICAM-1. Here we investigated the function of these adhesion molecules by binding and transendothelial invasion assays using a newly established bone marrow-derived endothelial cell line (BMEC). A combination of monoclonal antibodies against CD44 and VLA-4 significantly inhibited the adherence of B9/BM1 cells to BMEC and anti-CD44 mAb especially blocked B9/BM1 transendothelial invasion of unstimulated BMEC cells. Results of additional experiments, in which the cells were treated with anti-CD44 and hyaluronidase, demonstrated that the interaction of CD44 molecules on B9/BM1 cells with hyaluronan on BMEC cells was a critical factor in both adhesion and transendothelial invasion in this model. However, stimulation of BMEC with TNFα resulted in increased invasion by B9/BM1 cells, which was completely suppressed by anti-VCAM-1 mAb, implicating a significant role of this adhesion molecule in this process during inflammation.

Bone marrow metastatic myeloma cells promote osteoclastogenesis through RANKL on endothelial cells

We have been using the B9/BM1 murine bone marrow metastasis model to study the function of adhesion molecules in the cell-cell interactions and transendothelial migration, necessary for tumor metastasis. The cell surface phenotype of these cells, which colonize vertebral and femoral marrow after intravenous injection, shows great similarity to that of human myeloma cells. In the present study, we investigated the interaction between B9/BM1 cells and osteoclasts, which likely support tumor metastasis in bone marrow. We found that co-culturing B9/BM1 cells and bone marrow-derived endothelial cells (BMECs) in the presence of vitamin D3 and M-CSF promoted differentiation of primary osteoclast progenitors to osteoclasts (detected by TRAP staining), and that this effect was blocked when BMECs were separated from the other cells by a porous polycarbonate membrane. Flow cytometry analysis showed that BMECs expressed RANKL (receptor activator of NF-κB ligand) protein on their surface, and that this expression was up-regulated by co-culture with B9/BM1 cells. Accordingly, RT-PCR showed expression of RANKL mRNA also to be up-regulated in BMECs co-cultured with B9/BM1 cells. Addition of OPG (osteoprotegerin, a decoy RANKL receptor) to the co-culture system completely blocked osteoclast induction, as did addition of anti-CD44 antibody. Furthermore, intravenous injection of B9/BM1 cells substantially increased the numbers of TRAP-positive osteoclasts detected in mice in vivo. Taken together, these findings suggest that B9/BM1 myeloma cells act via CD44 to stimulate RANKL expression on BMECs, which in turn physically interact with osteoclast progenitors to promote their differentiation to osteoclasts and metastasis in bone marrow.

Platinum (II) complex with cyclometalating 2-phenylpyridine ligand showing high cytotoxicity against cisplatin-resistant cell.

A platinum (II) mononuclear complex with two kinds of 2-phenylpyridine which coordinate as cyclometalated and non-chelated ligands shows high cytotoxicity against cisplatin-resistant mouse sarcoma 180 cell in comparison with its related complexes.

Synthesis of a novel lipopeptide with α-melanocyte-stimulating hormone peptide ligand and its effect on liposome stability

Introduction of liposomes into target cells is important for drug delivery systems. For this purpose, the surface of the liposome is equipped with ligand peptides, which may bind to specific receptors on the cell membrane. An artificial novel lipopeptide (MSH-C4A2) containing the α-melanocyte-stimulating hormone (α-MSH) sequence and two long alkyl chains was designed and synthesized, and the liposome, composed of egg phosphatidylcholine (EPC) and MSH-C4A2, was prepared. The stability of the liposome was estimated by measuring calcein leakage from the liposome inner phase. The stability of the liposome decreased upon addition of MSH-A4C2, which seemed to be attributable to the amphiphilic property of the peptide moiety (α-MSH) of MSH-A2C4. The stability was, however, recovered fairly well upon addition of cholesterol (Ch) or phosphatidylglycerol (PG). It was concluded therefore that the ternary system, MSH-C4A2/Ch/EPC or MSH-C4A2/PG/EPC, is suitable for preparing the functional liposome.

Preparation of functional liposomes with peptide ligands and their binding to cell membranes

Two novel lipopeptides, which have the peptide ligands [α-melanocyte stimulating hormone (α-MSH)] sequence and repeated [Gly-Arg-Gly-Asp-Se (GRGDS) sequence[, are designed, synthesized by the solid-phase method, and introduced into liposome membranes by the freeze-thaw method. These liposomes bearing the peptide ligands on their surface are expected to bind to cell membranes. We have confirmed that the lipopeptides are introduced into liposome membranes almost quantitatively, while such a high degree of incorporation has not been accomplished in conventional methods. In this respect, the present method is superior to prepare surface-modified liposomes that are applicable to drug carriers and so on. We have also confirmed by using immunoelectron microscopy that the peptide ligands are actually located in an aqueous phase. It has been shown by flow cytometry that the liposome bearing α-MSH peptide ligand binds to B16 cells and the liposome bearing the repeated GRGDS sequence binds to NIH3T3 cells.

Solid-phase synthesis of dehydropeptide, AM-toxin II, using a novel selenyl linker by side-chain tethered strategy

Abstract The cyclic dehydrodepsipeptide, AM-toxin II, was efficiently synthesized by a solid-phase method using a novel selenyl linker. The cleavage from the resin with formation of a double bond is successfully achieved under mild oxidative conditions with TBHP.

67Zn and 1H NMR studies of Zn2+-imidazole and carboxylate complexes.

Abstract 67 Zn NMR studies of naturally abundant Zn 2+ -imidazole and carboxylate ligands Complexes are shown. Thus, quadrupolar relaxation changes in 67 Zn NMR caused by adding imidazole ligands are more remarkable than those by carboxylate ligands. The changes caused by adding less bulky imidazole ligands are more prominent than those caused by a bulky imidazole ligan. Changes in Zn 2+ quadrupolar relaxation rate caused by adding a cyclic hexapeptide consisting of L-histidine, L-cystein(Acm) and D-leucine are larger than those by a corresponding linear hexapeptide. Those changes in the quadrupolar relaxation rate of 67 Zn NMR among Zn 2+ complexes can be reasonably interpreted in terms of the differences of equilibrium constants of those complexes to a first approximation.

New selenyl linker for solid-phase synthesis of dehydropeptides

A novel linker possessing selenocyanate and masked carboxylic acid was developed for the solid-phase synthesis of dehydropeptides. This linker was used to demonstrate the synthesis of the model compound of RGD-conjugated dehydropeptide.

Functional nanoparticle based on β-cyclodextrin

Aminoethylcarbamoyl-β-cyclodextrin (AEC-β-CD)-based nanoparticle is prepared by an interfacial polyaddition reaction. The nanoparticle has high β-CD content and many amino groups on the surface. The β-CD cavity of this particle forms inclusion complexes with aromatic molecules, where the uptake ability significantly depends on physicochemical factors such as hydrophobicity, ionic charge, size, and shape of guest molecules.

Preparation, structure and cytotoxicity of cis-diammineplatinum(II) dinuclear complexes with 1-alkyluracil and imidate ligands ☆

Abstract A series of platinum dinuclear complexes with l-alkyluracil, cis -[Ph 2 (PtU) 2 (NH 3 ) 4 ] (NO 3 ) 2 , cis-[Pt 2 (n-BuU) 2 (NH 3 ) 4 ](NO 3 ) 2 , cis-[Ph 2 (BzlU) 2 (NH 3 ) 4 ](NO 3 ) 2 and cis-[Pt 2 (NaphCH 2 U) 2 (NH 3 ) 4 ](NO 3 ) 2 (head-to-head, where U=uracil), and with imide ligands, cis-[Ph 2 (SI 2 (NH 3 ) 4 ](NO 3 ) 2 (head-to-head), cis-[Pt 2 (DMGI) 2 (NH 3 ) 4 ](NO 3 ) 2 (head-to-head), cis-[Pt 2 (DMGI) 2 (NH 3 ) 4 ](NO 3 ) 2 (head-to-tail) and cis-[Pt 2 (DMGI) 2 (NH 3 ) 4 ] (NO 3 ) 2 (head-to-head, where SI = succinimidate, EMGI = 3-ethyl-3-methylglutarimidate and DMGi=3,3.dimethylglutarimidate) was synthesized, as well as platinum mononuelear complexes, cis-[PtCl(SI)(NH 3 ) 2 ] and cis- [Pt(SI) 2 (NH 3 ) 2 ]. The isomers of the dinuclear complexes (head-to-head and head-to-tail forms) were obtained separately by fractional recystallizations. Crystal structures of cis-[Pt 2 (SI) 2 (NH 3 ) 4 ](NO 3 ) 2 (head-to-head), cis-[Pt 2 (DMGI) 2 (NH 3 ) 4 ](NO 3 ) 2 (head-to-head) and cis-[Pt 2 (EMGI) 2 (NH 3 ) 4 ](NO 3 ) 2 (head-to-head) were determined by X-ray diffraction analysis. Cytotoxic activity was evaluated by the IC 50 value using mouse sarcoma S-180 cells. Most head-to-head complexes are considerably active, while the corresponding head-to-tail analogues are inactive. The active complexes, in general, release ligands in saline at 37°C, and a relationship between hydrophobicity of the complexes and the IC 50 value has been shown.