Makiya Nishikawa

Reactive oxygen species in tumor metastasis

Tumor metastasis is attributed not only to the abnormalities of cancer cells, but also to changes induced by the interaction of cancer cells and surrounding cells/tissues. The host immune response to cancer cells may contribute to an increased incidence of tumor metastasis. Surgical removal of tumor tissues can trigger tumor recurrence and metastatic tumor growth in distant organs. An important class of molecules involved in these events is the reactive oxygen species (ROS), which have been identified as involved in not only to tumor metastasis but also most disease processes. ROS will contribute to various aspects of malignant tumors, including carcinogenesis, aberrant growth, metastasis, and angiogenesis. High-level ROS, which can be reached by several anti-cancer treatments, suppresses tumor metastasis by destroying cancer cells because of the oxidative nature of the molecules. On the other hand, sublethal levels of ROS can induce additional changes in DNA of tumor cells to make those cells malignant, stimulate the proliferation of cancer cells, and activate the expression of various molecules, some of which assist cancer cells to form metastatic colonies. Thus, a precise understanding how ROS are generated and involved in tumor metastasis will help us to design better strategies to overcome such life-threatening events.

Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection

The development of exosomes as delivery vehicles requires understanding how and where exogenously administered exosomes are distributed in vivo. In the present study, we designed a fusion protein consisting of Gaussia luciferase and a truncated lactadherin, gLuc-lactadherin, and constructed a plasmid expressing the fusion protein. B16-BL6 murine melanoma cells were transfected with the plasmid, and exosomes released from the cells were collected by ultracentrifugation. Strong luciferase activity was detected in the fraction containing exosomes, indicating their efficient labeling with gLuc-lactadherin. Then, the labeled B16-BL6 exosomes were intravenously injected into mice, and their tissue distribution was evaluated. Pharmacokinetic analysis of the exosome blood concentration-time profile revealed that B16-BL6 exosomes disappeared very quickly from the blood circulation with a half-life of approximately 2min. Little luciferase activity was detected in the serum at 4h after exosome injection, suggesting rapid clearance of B16-BL6 exosomes in vivo. Moreover, sequential in vivo imaging revealed that the B16-BL6 exosome-derived signals distributed first to the liver and then to the lungs. These results indicate that gLuc-lactadherin labeling is useful for tracing exosomes in vivo and that B16-BL6 exosomes are rapidly cleared from the blood circulation after systemic administration.

Cell-specific delivery of genes with glycosylated carriers

Cationic liposomes and polymers have been accepted as effective non-viral vectors for gene delivery with low immunogenicity unlike viral vectors. However, the lack of organ or cell specificity sometimes hampers their application and the development of a cell-specific targeting technology for them attracts great interest in gene therapy. In this review, the potential of cell-specific delivery of genes with glycosylated liposomes or polymers is discussed. Galactosylated liposomes and poly(amino acids) are selectively taken up by the asialoglycoprotein receptor-positive liver parenchymal cells in vitro and in vivo after intravenous injection. DNA-galactosylated cationic liposome complexes show higher DNA uptake and gene expression in the liver parenchymal cells in vitro than DNA complexes with bare cationic liposomes. In the in vitro gene transfer experiment, galactosylated liposome complexes are more efficient than DNA-galactosylated poly(amino acids) complexes but they have some difficulties in their biodistribution control. On the other hand, introduction of mannose residues to carriers resulted in specific delivery of genes to non-parenchymal liver cells. These results suggest advantages of these glycosylated carriers in cell-specific targeted delivery of genes.

Hepatic uptake of polystyrene microspheres in rats: effect of particle size on intrahepatic distribution.

The in vivo disposition of polystyrene microsphere (MS) with the particle size of 50 nm (MS-50) or 500 nm (MS-500) was characterized after intravenous administration to rats. A rapid elimination from systemic circulation was observed for both MSs. Tissue distribution of MS-50 and MS-500 at 1 h after intravenous injection indicated that both MSs were exclusively distributed to liver and that small but significant amounts of MS-50 and MS-500 were also distributed to lung and spleen, respectively. To investigate the intrahepatic distribution of MS, liver was separated into liver parenchymal cells (PC) and non-parenchymal cells (NPC) at 1 or 6 h after intravenous administration. The contribution of each cell fraction was dependent on both the size of MS and the time after administration. Furthermore, by separating the NPC into endothelial cells and Kupffer cells using a centrifugal elutriation method, their contribution was also evaluated. For both MSs, Kupffer cells were recognized to be mostly responsible for the hepatic uptake, although a significant amount of MS-50 (about 28% of total uptake) was taken up by PC. On the other hand, there was little contribution of PC (about 5%) to the hepatic uptake of MS-500. The endothelial cells were contributed larger to the uptake of MS-500 (about 24%) than that of MS-50 (13%).

Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane: implication of a nonspecific process in efficient intracellular gene delivery

The mechanisms underlying the efficient gene transfer by a large‐volume and high‐speed intravenous injection of naked plasmid DNA (pDNA), a so‐called hydrodynamics‐based procedure, remain unclear and require further investigation. In this report, we have investigated possible mechanisms for the intracellular transport of naked pDNA by this procedure.

Nonviral vector-mediated RNA interference: its gene silencing characteristics and important factors to achieve RNAi-based gene therapy.

RNA interference (RNAi) is a potent and specific gene silencing event in which small interfering RNA (siRNA) degrades target mRNA. Therefore, RNAi is of potential use as a therapeutic approach for the treatment of a variety of diseases in which aberrant expression of mRNA causes a problem. RNAi can be achieved by delivering siRNA or vectors that transcribe siRNA or short-hairpin RNA (shRNA). The aim of this review is to examine the potential of nonviral vector-mediated RNAi technology in treating diseases. The characteristics of plasmid DNA expressing shRNA were compared with those of siRNA, focusing on the duration of gene silencing, delivery to target cells and target specificity. Recent progresses in prolonging the RNAi effect, improving the delivery to target cells and increasing the specificity of RNAi in vivo are also reviewed.

Biodegradable CpG DNA hydrogels for sustained delivery of doxorubicin and immunostimulatory signals in tumor-bearing mice.

Immunostimulatory CpG DNA was self-assembled to form DNA hydrogels for use as a sustained delivery system for both intercalated doxorubicin (DXR) and immunostimulatory CpG motifs for cancer treatment. X-shaped DNA (X-DNA) was designed as a building unit, and underwent ligation to form DNA hydrogels. Two types of X-DNA were constructed using four oligodeoxynucleotides each, one containing six potent CpG motifs (CpG X-DNA) and the other with none (CpG-free X-DNA). CpG X-DNA was more effective than its components or the CpG-free counterpart in terms of the production of tumor necrosis factor-α from murine macrophage-like RAW264.7 cells, as well as maturation of the murine dendritic DC2.4 cells. The cytotoxic effects of X-DNA, DXR and their complexes were examined in a co-culture system of colon26/Luc cells, a murine adenocarcinoma clone stably expressing firefly luciferase, and RAW264.7 cells. DXR/CpG X-DNA showed the highest ability to inhibit the proliferation of colon26/Luc cells. DXR was slowly released from CpG DNA hydrogels. Injections of DXR/CpG DNA hydrogels into a subcutaneous colon26 tumor effectively inhibited tumor growth. These results show that CpG DNA hydrogels are an effective sustained system for delivery of immunostimulatory signals to TLR9-positive immune cells and DXR to cancer cells.

Macrophage activation by a DNA/cationic liposome complex requires endosomal acidification and TLR9‐dependent and ‐independent pathways

Previously, we showed that bacterial DNA and vertebrate DNA/cationic liposome complexes stimulate potent inflammatory responses in cultured mouse macrophages. In the present study, we examined whether endocytosis and subsequent acidification are associated with these responses. The endocytosis inhibitor, cytochalasin B, reduced tmor necrosis factor α (TNF‐α) production by a plasmid DNA (pDNA)/cationic liposome complex. The endosomal acidification inhibitor, monensin, inhibited cytokine production by pDNA or a calf thymus DNA/liposome complex. These results suggest, similarly to CpG motif‐dependent responses, that endocytosis and subsequent endosomal acidification are also required for these inflammatory responses. It is intriguing that another inhibitor of endosomal acidification, bafilomycin A, stimulated the production of TNF‐α mRNA and its protein after removal of the pDNA/liposome complex and inhibitors, although it inhibited the release of interleukin‐6. Similar phenomena were observed in the activation of macrophages by CpG oligodeoxynucleotide, calf thymus DNA, and Escherichia coli DNA complexed with liposomes. Moreover, bafilomycin A also induced a high degree of TNF‐α release after stimulation with naked pDNA. These results suggest that bafilomycin A increases TNF‐α production induced by DNA at the transcriptional level via an as‐yet unknown mechanism. Furthermore, we investigated the contribution of Toll‐like receptor 9 (TLR9), the receptor of CpG motifs, to the cell activation by the DNA/cationic liposome complex using the macrophages from TLR9−/− mice. We observed a reduced inflammatory cytokine release from macrophages of TLR9−/− mice compared with wild‐type mice. However, the cytokine production was not completely abolished, suggesting that the DNA/cationic liposome complex can induce macrophage activation via TLR9‐dependent and ‐independent pathways.

Synthesis and Pharmacokinetics of a New Liver-Specific Carrier, Glycosylated Carboxymethyl-Dextran, and Its Application to Drug Targeting

To develop a new carrier system for hepatic targeting, carboxymethyl-dextran (CMD) was modified with galactose and mannose residues (Gal-CMD, Man-CMD), and their disposition characteristics were studied in mice using 14C-labeled dextran. At a dose of 1 mg/kg, i.v.-injected Gal-CMD and Man-CMD rapidly accumulated in the liver parenchymal and nonparenchymal cells, respectively, because of their preferential uptake via carbohydrate receptors in these cells. Pharmacokinetic analysis revealed that their uptake rates were sufficiently large for selective drug targeting. Targeting of cytosine β-D-arabinoside (araC) was studied using Gal-CMD as a specific carrier to the hepatocytes. From the conjugate of araC with Gal-CMD, araC was released with a half-life of 36 hr in phosphate buffer (pH 7.4) and 23 hr in plasma. An in vivo biodistribution study demonstrated a disposition profile of the conjugated araC similar to that of the carrier, and selective delivery to hepatocytes of up to 80% of the dose was achieved. These findings suggest that glycosylated CMDs are carriers with a high affinity to liver parenchymal or nonparenchymal cells without any affinity to other tissues.

Design and development of nanosized DNA assemblies in polypod-like structures as efficient vehicles for immunostimulatory CpG motifs to immune cells.

The immunostimulatory activity of phosphodiester DNA containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides, or CpG motifs, was significantly increased by the formation of Y-, X-, or dendrimer-like multibranched shape. These results suggest the possibility that the activity of CpG DNA is a function of the structural properties of branched DNA assemblies. To elucidate the relationship between them, we have designed and developed nanosized DNA assemblies in polypod-like structures (polypod-like structured DNA, or polypodna for short) using oligodeoxynucleotides (ODNs) containing CpG motifs and investigated their structural and immunological properties. Those assemblies consisting of three (tripodna) to eight (octapodna) ODNs were successfully obtained, but one consisting of 12 ODNs was not when 36-mer ODNs were annealed under physiological sodium chloride concentration. High-speed atomic force microscopy revealed that these assemblies were in polypod-like structures. The apparent size of the products was about 10 nm in diameter, and there was an increasing trend with an increase in ODN length or with the pod number. Circular dichroism spectral data showed that DNA in polypodna preparations were in the B-form. The melting temperature of polypodna decreased with increasing pod number. Each polypodna induced the secretion of tumor necrosis factor-α and interleukin-6 from macrophage-like RAW264.7 cells, with the greatest induction by those with hexa- and octapodna. Increasing the pod number increased the uptake by RAW264.7 cells but reduced the stability in serum. These results indicate that CpG DNA-containing polypodna preparations with six or more pods are a promising nanosized device with biodegradability and high immunostimulatory activity.