Hiroaki Kinoh

MEMBRANE TYPE 4 MATRIX METALLOPROTEINASE (MT4-MMP, MMP-17) IS A GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEINASE

Among the five membrane-type matrix metalloproteinases (MT-MMPs), MT1-, MT2-, MT3-, and MT5-MMPs have about a 20-amino acid cytoplasmic tail following the transmembrane domain. In contrast, a putative transmembrane domain of MT4-MMP locates at the very C-terminal end, and the expected cytoplasmic tail is very short or nonexistent. Such sequences often act as a glycosylphosphatidylinositol (GPI) anchoring signal rather than as a transmembrane domain. We thus examined the possibility that MT4-MMP is a GPI-anchored proteinase. Our results showed that [3H]ethanolamine, which can be incorporated into the GPI unit, specifically labeled the MT4-MMP C-terminal end in a sequence-dependent manner. In addition, phosphatidylinositol-specific phospholipase C treatment released the MT4-MMP from the surface of transfected cells. These results indicate that MT4-MMP is the first GPI-anchored proteinase in the MMP family. During cultivation of the transfected cells, MT4-MMP appeared to be shed from the cell surface by the action of an endogenous metalloproteinase. GPI anchoring of MT4-MMP on the cell surface indicates a unique biological function and character for this proteinase.

Absence of mechanical allodynia and Aβ‐fiber sprouting after sciatic nerve injury in mice lacking membrane‐type 5 matrix metalloproteinase

Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade extracellular matrix components. Membrane‐type 5 MMP (MT5‐MMP/MMP‐24) was identified as neuron‐specific, and is believed to contribute to neuronal circuit formation and plasticity. To elucidate its function in vivo, we have generated mice lacking MT5‐MMP by gene targeting. MT5‐MMP‐deficient mice were born without obvious morphological abnormalities. No apparent histological defects were observed in the nervous system either. However, MT5‐MMP‐deficient mice did not develop neuropathic pain with mechanical allodynia after sciatic nerve injury, though responses to acute noxious stimuli were normal. Neuropathic pain induced by peripheral nerve lesions is known to accompany structural reorganization of the nervous system. Intraneural injection of cholera toxin B subunit, a transganglionic tracer, into the injured sciatic nerve of wild‐type mice revealed that the myelinated Aβ‐fiber primary afferents sprouted from laminae III–VI of the dorsal horn of the spinal cord and invaded lamina II. However, no such sprouting and invasion of Aβ‐fibers were observed in MT5‐MMP‐deficient mice. These findings suggest that MT5‐MMP is essential for the development of mechanical allodynia and plays an important role in neuronal plasticity in this mouse model.

Human membrane type-4 matrix metalloproteinase (MT4-MMP) is encoded by a novel major transcript: isolation of complementary DNA clones for human and mouse mt4-mmp transcripts

Five distinct membrane‐type matrix metalloproteinases (MT‐MMP) have been reported by cDNA cloning. However, the mt4‐mmp gene product (MMP‐17) has not been identified yet in spite of the cDNA isolation [Puente et al. (1996), Cancer Res. 56, 944–949]. In this study, we re‐examined the transcripts for human mt4‐mmp by 5′ RACE and identified two types of transcripts. The minor one corresponded to the cDNA reported by Puente et al. and failed to express protein, and the other is the major transcript that has an extended open reading frame and expressed 67 and 71 kDa translation products. Thus, functional mt4‐mmp has been identified for the first time.

Generation of a recombinant Sendai virus that is selectively activated and lyses human tumor cells expressing matrix metalloproteinases

Malignant tumor cells often express matrix metalloproteinases (MMPs) at a high level to enable their dissemination and metastasis. Sendai virus (SeV), a nonsegmented negative strand RNA virus, spreads in the target tissues in vivo via cleavage activation of the viral fusion glycoprotein by a tissue-specific, trypsin-like enzyme. By deleting the viral matrix protein, we previously generated a recombinant SeV that does not bud to mature virions, but is highly fusogenic and spreads extensively from cell to cell in a trypsin-dependent manner. Here, we changed the tryptic cleavage site of the fusion glycoprotein of this virus to a site susceptible to MMPs. The resulting recombinant virus was no longer activated by trypsin but spread efficiently in cultured cells supplemented with MMP2 or MMP9 and in human tumor cell lines expressing these MMPs. Furthermore, the virus spread extensively in tumor cells xenotrasplanted to nude mice without disseminating to the surrounding normal cells, leading to the inhibition of the tumor growth in the mice. These results demonstrate the selective targeting and killing of human tumor cells by recombinant SeV technology and greatly advance the reemerging concept of oncolytic virotherapy, which currently appears to rely largely upon a natural preference of certain viruses for cancer cells.

Selective intracellular delivery of proteasome inhibitors through pH-sensitive polymeric micelles directed to efficient antitumor therapy.

The ubiquitin-proteasome system is central in the regulation of cellular proteins controlling cell cycle progression and apoptosis, drawing much interest for developing effective targeted cancer therapies. Herein, we developed a novel pH-responsive polymeric-micelle-based carrier system to effectively deliver the proteasome inhibitor MG132 into cancer cells. MG132 is covalently bound to the block copolymer composed of polyethylene glycol (PEG) and polyaspartate through an acid-labile hydrazone bond. This bond is stable at physiological condition, but hydrolytically degradable in acidic compartments in the cell, such as late-endosomes and lysosomes, and thus, it was used for controlled release of MG132 after EPR-mediated preferential accumulation of the micelles into the tumor. MG132-loaded micelles have monodispersed size distribution with an average diameter of 45nm, and critical micelle concentration is well below 10(-7)M. In vitro studies against several cancer cell lines confirmed that MG132-loaded micelles retained the cytotoxic effect, and this activity was indeed due to the inhibition of proteasome by released MG132 from the micelles. Real-time in vitro confocal-microscopy experiments clearly indicated that MG132-conjugated micelles disintegrated only inside the target cells. By intravital confocal micro-videography, we also confirmed the prolonged circulation of MG132 loaded micelles in the bloodstream, which lead to tumor specific accumulation of micelles, as confirmed by in vivo imaging 24h after injection. These micelles showed significantly lower in vivo toxicity than free MG132, while achieving remarkable antitumor effect against a subcutaneous HeLa-luc tumor model. Our findings create a paradigm for future development of polymeric-micelle-based carrier system for other peptide aldehyde type proteasome inhibitors to make them effective cohort of the existing cancer therapeutic regiments.

Purification and Characterization of the Egg Jelly Macromolecules, Sialoglycoprotein and Fucose Sulfate Glycoconjugate, of the Sea Urchin Hemicentrotus Pulcherrimus

A sialoglycoprotein and a fucose sulfate glycoconjugate (FSG) were purified from egg jelly of the sea urchin Hemicentrotus pulcherrimus. Sialoglycoprotein which consisted of sialic acid (90%, w/w) and protein (10%, w/w) did not cause induction of the acrosome reaction and sperm isoagglutination. FSG which contained one mol sulfate/mol fucose possessed 2.0 times protein to fucose by weight. The proteins in intact FSG were separated to two major (258 kDa and 237 kDa) and one minor (120 kDa) proteins by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) in the presence of 2‐mercaptoethanol (2‐ME) while the proteins could not be separated by HPLC in the presence of 0.1% SDS or SDS‐PAGE without 2‐ME. However, after carboxymethylation of FSG, two major (260 kDa and 240 kDa) proteins and two minor (140 kDa and 135 kDa) proteins were separated from the fucose sulfate moiety by HPLC in the presence of 0.1% SDS or SDS‐PAGE without 2‐ME. When FSG was first carboxymethylated with non‐radioactive iodoacetic acid and then reduced with 2‐ME and finally carboxymethylated with 14C‐iodoacetic acid, the most of radioactivity was detected in 140 kDa and 135 kDa proteins. Carboxymethylted‐FSG was less potent than intact FSG in induction of the acrosome reaction. Fucoidan, a fucose sulfate polymer, did not induce the acrosome reaction.

Recombinant Sendai Virus Vector Induces Complete Remission of Established Brain Tumors through Efficient Interleukin-2 Gene Transfer in Vaccinated Rats

Purpose: Sendai virus (SeV), a murine parainfluenza virus type I, replicates independent of cellular genome and directs high-level gene expressions when used as a viral vector. We constructed a nontransmissible recombinant SeV vector by deleting the matrix (M) and fusion (F) genes from its genome (SeV/ΔMΔF) to enhance its safety. We also estimated the therapeutic efficacy of the novel vector system against a rat glioblastoma model. Experimental Design: We administered the recombinant SeV vector carrying the lacZ gene or the human interleukin-2 (hIL-2) gene into established 9L brain tumors in vivo simultaneous with peripheral vaccination using irradiated 9L cells. Sequential monitoring with magnetic resonance imaging was used to evaluate the therapeutic efficacy. Results: We found extensive transduction of the lacZ gene into the brain tumors and confirmed sufficient amounts of interleukin 2 (IL-2) production by hIL2-SeV/ΔMΔF both in vitro and in vivo. The magnetic resonance imaging study showed that the intracerebral injection of hIL2-SeV/ΔMΔF brought about significant reduction of the tumor growth, including complete elimination of the established brain tumors. The 51Cr release assay showed that significant amounts of 9L-specific cytotoxic T cells were induced by the peripheral vaccination. Immunohistochemical analysis revealed that CD4+ T cells and CD8+ T cells were abundantly infiltrated in the target tumors. Conclusion: The present results show that the recombinant nontransmissible SeV vector provides efficient in vivo gene transfer that induces significant regression of the established brain tumors and suggest that it will be a safe and useful viral vector for the clinical practice of glioma gene therapy.

Sustained and NK/CD4+ T Cell-Dependent Efficient Prevention of Lung Metastasis Induced by Dendritic Cells Harboring Recombinant Sendai Virus

We recently demonstrated efficient antitumor immunity against murine tumors using dendritic cells (DCs) activated by recombinant Sendai viruses (rSeVs), and proposed a new concept, “immunostimulatory virotherapy,” for cancer immunotherapy. However, there has been little information on the efficacy of this method in preventing metastatic diseases. In this study, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV (rSeV/dF) using a murine model of lung metastasis. Bolus and i.v. administration of DCs harboring rSeV/dF-expressing GFP without pulsation of tumor Ag (DC-rSeV/dF-GFP) 2 days before tumor inoculation showed efficient prevention against lung metastasis of c1300 neuroblastoma, but not of RM-9 prostatic cancer. We found that the timing of DC therapy was critical for the inhibition of pulmonary metastasis of RM-9, and that the optimal effect of DCs was seen 28 days before tumor inoculation. Interestingly, the antimetastatic effect was sustained for over 3 mo, even when administered DCs were already cleared from the lung and organs related to the immune system. Although NK cell activity had already declined to baseline at the time of tumor inoculation, Ab-mediated depletion studies revealed that CD4+ cells as well as the presence of, but not the activation of, NK cells were crucial to the prevention of lung metastasis. These results are the first demonstration of efficient inhibition of lung metastasis via bolus administration of virally activated DCs that was sustained and NK/CD4+ cell-dependent, and may suggest a potentially new mechanism of DC-based immunotherapy for advanced malignancies.

Generation of optimized and urokinase-targeted oncolytic Sendai virus vectors applicable for various human malignancies

We previously reported the development of a prototype ‘oncolytic Sendai virus (SeV) vector’ formed by introducing two major genomic modifications to the original SeV, namely deletion of the matrix (M) gene to avoid budding of secondary viral particles and manipulation of the trypsin-dependent cleavage site of the fusion (F) gene to generate protease-specific sequences. As a result, the ‘oncolytic SeV’ that was susceptible to matrix metalloproteinases (MMPs) was shown to selectively kill MMP-expressing tumors through syncytium formation in vitro and in vivo. However, its efficacy has been relatively limited because of the requirement of higher expression of MMPs and smaller populations of MMP-expressing tumors. To overcome these limitations, we have designed an optimized and dramatically powerful oncolytic SeV vector. Truncation of 14-amino acid residues of the cytoplasmic domain of F protein resulted in dramatic enhancement of cell-killing activities of oncolytic SeV, and the combination with replacement of the trypsin cleavage site with the new urokinase type plasminogen activator (uPA)-sensitive sequence (SGRS) led a variety of human tumors, including prostate (PC-3), renal (CAKI-I), pancreatic (BxPC3) and lung (PC14) cancers, to extensive death through massive cell-to-cell spreading without significant dissemination to the surrounding noncancerous tissue in vivo. These results indicate a dramatic improvement of antitumor activity; therefore, extensive utility of the newly designed uPA-targeted oncolytic SeV has significant potential for treating patients bearing urokinase-expressing cancers in clinical settings.

Polymeric micelles loaded with platinum anticancer drugs target preangiogenic micrometastatic niches associated with inflammation

Nanocarriers have been used for specific delivery of therapeutic agents to solid tumors based on the enhanced permeability and retention in cancerous tissues. Despite metastasis is the main reason of cancer-related death and a priority for nanocarrier-based therapies, the targeting ability of nanocarriers to the metastatic disease is poorly understood, especially for preangiogenic micrometastases as nanocarriers usually use the malignant neovasculature for enhancing their accumulation. Thus, herein, we studied the ability of micellar nanocarriers incorporating (1,2-diaminocyclohexane)platinum(II) (DACHPt) for treating liver metastases of bioluminescent murine colon adenocarcinoma C-26, during overt and preangiogenic metastatic stages. After intravenous injection, DACHPt-loaded micelles (DACHPt/m) effectively inhibited the tumor growth in both metastatic tumor models. While the anticancer activity of the micelles against overt metastases was associated with their selective accumulation in cancerous tissues having neovasculature, the ability of DACHPt/m to target preangiogenic metastases was correlated with the inflammatory microenvironment of the niche. This targeting capability of polymeric micelles to preangiogenic metastasis may provide a novel approach for early diagnosis and treatment of metastases.