研究者総覧
内堀 亮介 (ｳﾁﾎﾞﾘ ﾘｮｳｽｹ)
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Last Updated :2023/05/31
研究活動情報
論文
- Ryosuke Uchibori, Tomonori Tsukahara, Ken Ohmine, Keiya OzawaINTERNATIONAL JOURNAL OF HEMATOLOGY 99 4 377 - 382 2014年04月 [査読有り][通常論文]
Cellular and gene therapies represent promising treatment strategies at the frontier of medicine. Hematopoietic stem cells, lymphocytes, and mesenchymal stem cells (MSCs) can all serve as sources of cells for use in such therapies. Strategies for gene therapy are often based on those of cell therapy, and it is anticipated that some examples will be put to practical use in the near future. Given their ability to support hematopoiesis, MSCs may be useful for the enhancement of stem cell engraftment, and the acceleration of hematopoietic reconstitution. Furthermore, MSCs may advance the treatment of severe graft-versus-host disease, based on their immunosuppressive ability. This application is also based on the homing behavior of MSCs to sites of injury and inflammation. Interestingly, MSCs possess tumor-homing ability, opening up the possibility of applications in the targeted delivery of anti-cancer genes to tumors. Many reports have indicated that MSCs can be utilized to target tumors and to deliver anti-cancer molecules locally, as tumors are recognized as non-healing wounds with inflammatory tissue. Here, we review both the potential of MSCs as cellular vehicles for targeted cancer therapy and the molecular mechanisms underlying MSC accumulation at tumor sites. - Novel anti-tumor mechanism of galanin receptor type 2 in head and neck squamous cell carcinoma cellsTakayuki Uehara, Takeharu Kanazawa, Hiroaki Mizukami, Ryosuke Uchibori, Tomonori Tsukahara, Masashi Urabe, Akihiro Kume, Kiyoshi Misawa, Thomas E. Carey, Mikio Suzuki, Keiichi Ichimura, Keiya OzawaCancer Science 105 1 72 - 80 2014年01月 [査読有り][通常論文]
Galanin and its receptors, GALR1 and GALR2, are known tumor suppressors and potential therapeutic targets in head and neck squamous cell carcinoma (HNSCC). Previously, we demonstrated that, in GALR1-expressing HNSCC cells, the addition of galanin suppressed tumor proliferation via upregulation of ERK1/2 and cyclin-dependent kinase inhibitors, whereas, in GALR2-expressing cells, the addition of galanin not only suppressed proliferation, but also induced apoptosis. In this study, we first transduced HEp-2 and KB cell lines using a recombinant adeno-associated virus (rAAV)-green fluorescent protein (GFP) vector and confirmed a high GFP expression rate (> 90%) in both cell lines at the standard vector dose. Next, we demonstrated that GALR2 expression in the presence of galanin suppressed cell viability to 40-60% after 72 h in both cell lines. Additionally, the annexin V-positive rate and sub-G0/G1 phase population were significantly elevated in HEp-2 cells (mock vs GALR2: 12.3 vs 25.0% (P < 0.01) and 9.1 vs 32.0% (P < 0.05), respectively) after 48 h. These changes were also observed in KB cells, although to a lesser extent. Furthermore, in HEp-2 cells, GALR2-mediated apoptosis was caspase-independent, involving downregulation of ERK1/2, followed by induction of the pro-apoptotic Bcl-2 protein, Bim. These results illustrate that transient GALR2 expression in the presence of galanin induces apoptosis via diverse pathways and serves as a platform for suicide gene therapy against HNSCC. © 2013 The Authors. - Tomonori Tsukahara, Ken Ohmine, Chihiro Yamamoto, Ryosuke Uchibori, Hiroyuki Ido, Takeshi Teruya, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Masataka Nakamura, Junichi Mineno, Kazutoh Takesako, Isabelle Riviere, Michel Sadelain, Renier Brentjens, Keiya OzawaBIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 438 1 84 - 89 2013年08月 [査読有り][通常論文]
Adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) is promising for treatment of advanced B-cell malignancies. Tumor targeting of CAR-modified T-cells is likely to contribute therapeutic potency; therefore we examined the relationship between the ability of CD19-specific CAR (CD19-CAR)-transduced T-cells to accumulate at CD19(+) tumor lesions, and their ability to provide antitumor effects in xenograft mouse models. Normal human peripheral blood lymphocytes, activated with immobilized RetroNectin and anti-CD3 antibodies, were transduced with retroviral vectors that encode CD19-CAR. Expanded CD19-CAR T-cells with a high transgene expression level of about 75% produced IL-2 and IFN-gamma in response to CD19, and lysed both Raji and Daudi CD19(+) human B-cell lymphoma cell lines. Furthermore, these cells efficiently accumulated at Raji tumor lesions where they suppressed tumor progression and prolonged survival in tumor-bearing Rag2(-/-)gamma c(-/-) immunodeficient mice compared to control cohorts. These results show that the ability of CD19-CAR T-cells to home in on tumor lesions is pivotal for their anti-tumor effects in our xenograft models, and therefore may enhance the efficacy of adoptive T-cell therapy for refractory B-cell lymphoma. (C) 2013 Elsevier Inc. All rights reserved. - Uchibori R, Tsukahara T, Mizuguchi H, Saga Y, Urabe M, Mizukami H, Kume A, Ozawa KCancer research 73 1 364 - 372 1 2013年01月 [査読有り][通常論文]
Mesenchymal stem cells (MSC) accumulate at tumor sites when injected into tumor-bearing mice, perhaps offering cellular vectors for cancer-targeted gene therapy. However, the molecular mechanisms involved in MSC targeting the tumors are presently little understood. We focused on MSC-endothelial cell (EC) adhesion following TNF-alpha stimulation in an attempt to elucidate these mechanisms. Interestingly, stimulation of MSCs with TNF-alpha enhanced the adhesion of MSCs to endothelial cells in vitro. This adhesion was partially inhibited by blocking antibodies against vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4). It is well known that TNF-alpha induces VCAM-1 expression via the NF-kappa B signaling pathway. Parthenolide has an anti-inflammatory activity and suppressed NF-kappa B activity by inhibition of I kappa B alpha phosphorylation after TNF-a stimulation and strongly inhibited TNF-alpha-induced VCAM-1 expression on MSCs. In vivo imaging using luciferase-expressing MSCs revealed that the bioluminescent signal gradually increased at tumor sites in mice injected with untreated MSCs. In contrast, we observed very weak signals at tumor sites in mice injected with parthenolide-treated MSCs. Our results suggest that NF-kappa B activity regulates MSC accumulation at tumors, by inducing VCAM-1 and thereby its interaction with tumor vessel endothelial cells. These findings have implications for the ongoing development of efficient MSC-based gene therapies for cancer treatment. Cancer Res; 73(1); 364-72. (C)2012 AACR. - Uchibori R, Ozawa K[Rinsho ketsueki] The Japanese journal of clinical hematology 51 11 1641 - 1646 11 2010年11月 [査読有り][通常論文]
- Minoru Kobayashi, Takashi Murakami, Ryosuke Uchibori, Nicole A. L. Chun, Eiji Kobayashi, Tatsuo Morita, Keiya OzawaJOURNAL OF UROLOGY 183 5 2029 - 2035 2010年05月 [査読有り][通常論文]
Purpose: Since renal cell carcinoma is considered an immunogenic tumor, testing therapeutic strategies has been impeded by the lack of relevant tumor models in immunocompetent animals. Recent advances in bioluminescence imaging permit sensitive in vivo detection and quantification of cells emitting light. Thus, we established bioluminescent rat renal cell carcinoma cell lines for immunocompetent rats. Materials and Methods: The rat renal cell carcinoma cell line ACI-RCC stemming from chemically induced renal cell carcinoma in syngeneic ACI rats was stably transfected with a recombinant retroviral vector encoding luciferase genes derived from fireflies (ACI-RCC-ffLuc) or click beetles (ACI-RCC-cbLuc). Cell line growth patterns were characterized by bioluminescence imaging. Results: Linear correlations noted observed between cell number and photon counts in each cell type. ACI-RCC-cbLuc emitted light about 500-fold higher than ACI-RCC-ffLuc. When transplanted subcutaneously, only ACI-RCC-ffLuc grew, possibly because of less antigenicity. ACI-RCC-ffLuc photon emission correlated significantly with subcutaneous tumor size. Orthotopic tumor growth and subsequent metastatic spread were monitored with time by increased photon intensity on bioluminescence imaging. Based on ACI-RCC-cbLuc bioluminescent intensity the in vitro screening test allowed the identification of several anticancer agents, including molecules related to human renal cell carcinoma progression. Conclusions: The new in vivo rat renal cell carcinoma model with luciferase labeled tumor cells allowed us to monitor tumor growth noninvasively and semiquantitatively by bioluminescence imaging. This model system coupled with in vitro screening permits precise evaluation of tumor behavior in intact animals and determination of the therapeutic efficacy of anticancer agents for renal cell carcinoma. - Okada T, Nonaka-Sarukawa M, Uchibori R, Kinoshita K, Hayashita-Kinoh H, Nitahara-Kasahara Y, Takeda S, Ozawa KHuman gene therapy 20 1013 - 1021 2009年09月 [査読有り][通常論文]
- Ryosuke Uchibori, Takashi Okada, Takayuki Ito, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Keiya OzawaJOURNAL OF GENE MEDICINE 11 5 373 - 381 2009年05月 [査読有り][通常論文]
Background Mesenchymal stein cells (MSCs) are a promising vehicle for targeted cancer gene therapy because of their potential of tumor tropism. For efficient therapeutic application, we developed retroviral vector-producing MSCs that enhance tumor transduction via progeny vector production. Methods Rat bone marrow-derived MSCs were nucleofected with the proviral plasmids (vesicular stomatitis virus-G protein-pseudotyped retroviral vector components) (VP-MSCs) or pLTR plasmid alone (non-VP-MSCs). The luciferase-based in vivo imaging system was used to assess gene expression periodically. To evaluate the anticancer effects, we administered MSCs expressing herpes simplex virus-thymidine kinase (HSV-tk) into the left ventricular cavity of nude mice engrafted with 9L glioma cells subcutaneously. Results In vivo imaging revealed that administration of luciferase-expressing non-VP-MSCs enhanced the bioluminescence signal at the inoculation sites of 9L cells, whereas no accumulation was observed in juice at the site of the control Rat-1 fibroblasts. Compared to non-VP-MSCs, the administration of VP-MSCs resulted in significant augmentation of the signal with an increase in transgene copy number. Immunohistochemical analysis showed marked luciferase expression at the tumor periphery in mice injected with VP-MSCs, whereas little expression was detected in those in injected. with non-VP-MSCs. Under the continuous infusion of ganciclovir systemic, administration of VP-MSCs expressing HSV-tk Suppressed tumor growth more effectively than non-VP-MSC administration, whereas no anticancer effect was observed Without ganciclovir treatment. Furthermore, VP-MSC administration caused no transgene transduction in the normal tissues and organs. Conclusions VP-MSCs accumulated at the site of tumors after intravascular injection in tumor-bearing mice, followed by in situ gene transfer to tumors without transduction of normal organs. When applied to the HSV-tk/ganciclovir suicide gene therapy, more efficient tumor growth Suppression was observed using VP-MSCs compared to non-VP-MSCs. This VP-MSC-based system has great potential for improved cancer gene therapy. Copyright (C) 2009 John Wiley & Sons, Ltd. - Keiya Ozawa, Kazuya Sato, Iekuni Oh, Katsutoshi Ozaki, Ryosuke Uchibori, Yoko Obara, Yuji Kikuchi, Takayuki Ito, Takashi Okada, Masashi Urabe, Hiroaki Mizukami, Akihiro KumeJOURNAL OF AUTOIMMUNITY 30 3 121 - 127 2008年05月 [査読有り][通常論文]
Mesenchymal stem cells (MSCs) are considered to be a promising platform for cell and gene therapy for a variety of diseases. First, in the field of hematopoietic stem cell transplantation, there are two applications of MSCs: 1) the improvement of stem cell engrafting and the acceleration of hematopoietic reconstitution based on the hematopoiesis-supporting ability; and 2) the treatment of severe graft-versus-host disease (GVHD) based on the immunomodulatory ability. Regarding the immunosuppressive ability, we found that nitric oxide (NO) is involved in the MSC-mediated suppression of T cell proliferation. Second, tumor-bearing nude mice were injected with luciferase-expressing MSCs. An in vivo imaging analysis showed the significant accumulation of the MSCs at the site of tumors. The findings suggest that MSCs can be utilized to target metastatic tumors and to deliver anti-cancer molecules locally. As the third application, MSCs may be utilized as a cellular vehicle for protein-supplement gene therapy. When long-term transgene expression is needed, a therapeutic gene should be introduced with a minimal risk of insertional mutagenesis. To this end, site-specific integration into the AAVS 1 locus on the chromosome 19 (19q13.4) by using the integration machinery of adeno-associated virus (AAV) would be particularly valuable. There will be wide-ranging applications of MSCs to frontier medical treatments in the near future. (C) 2008 Elsevier Ltd. All rights reserved. - Takayuki Ito, Takashi Okada, Jun Mimuro, Hiroshi Miyashita, Ryosuke Uchibori, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Masafumi Takahashi, Uichi Ikeda, Yoichi Sakata, Kazuyuki Shimada, Keiya OzawaHYPERTENSION 50 3 531 - 536 2007年09月 [査読有り][通常論文]
Prostacyclin synthase (PGIS) is the final committed enzyme in the metabolic pathway of prostacyclin production. The therapeutic option of intravenous prostacyclin infusion in patients with pulmonary arterial hypertension is limited by the short half-life of the drug and life-threatening catheter-related complications. To develop a better delivery system for prostacyclin, we examined the feasibility of intramuscular injection of an adenoassociated virus (AAV) vector expressing PGIS for preventing monocrotaline-induced pulmonary arterial hypertension in rats. We developed an AAV serotype 1-based vector carrying a human PGIS gene (AAV-PGIS). AAV-PGIS or the control AAV vector expressing enhanced green fluorescent protein was injected into the anterior tibial muscles of 3-week-old male Wistar rats; this was followed by the monocrotaline administration at 7 weeks. Eight weeks after injecting the vector, the plasma levels of 6-keto-prostaglandin F-1 alpha increased in a vector dose-dependent manner. At this time point, the PGIS transduction (1x10(10) genome copies per body) significantly decreased mean pulmonary arterial pressure (33.9 +/- 2.4 versus 46.1 +/- 3.0 mm Hg; P < 0.05), pulmonary vascular resistance (0.26 +/- 0.03 versus 0.41 +/- 0.03 mm Hg . mL(-1) . min(-1) . kg(-1); P < 0.05), and medial thickness of the peripheral pulmonary artery (14.6 +/- 1.5% versus 23.5 +/- 0.5%; P < 0.01) as compared with the controls. Furthermore, the PGIS-transduced rats demonstrated significantly improved survival rates as compared with the controls (100% versus 50%; P < 0.05) at 8 weeks postmonocrotaline administration. An intramuscular injection of AAV-PGIS prevents monocrotaline-pulmonary arterial hypertension in rats and provides a new therapeutic alternative for preventing pulmonary arterial hypertension in humans. - Takayuki Ito, Takashi Okada, Hiroshi Miyashita, Tatsuya Nomoto, Mutsuko Nonaka-Sarukawa, Ryosuke Uchibori, Yoshikazu Maeda, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Masafumi Takahashi, Uichi Ikeda, Kazuyuki Shimada, Keiya OzawaCIRCULATION RESEARCH 101 7 734 - 741 2007年09月 [査読有り][通常論文]
Pulmonary arterial hypertension (PAH) is a fatal disease associated with inflammation and pathological remodeling of the pulmonary artery (PA). Interleukin (IL)-10 is a pleiotropic antiinflammatory cytokine with vasculoprotective properties. Here, we report the preventive effects of IL-10 on monocrotaline-induced PAH. Three-week-old Wistar rats were intramuscularly injected with an adeno-associated virus serotype 1 vector expressing IL-10, followed by monocrotaline injection at 7 weeks old. IL-10 transduction significantly improved survival rates of the PAH rats 8 weeks after monocrotaline administration compared with control gene transduction (75% versus 0%, P < 0.01). IL-10 also significantly reduced mean PA pressure (22.8 +/- 1.5 versus 29.7 +/- 2.8 mm Hg, P < 0.05), a weight ratio of right ventricle to left ventricle plus septum (0.35 +/- 0.04 versus 0.42 +/- 0.05, P < 0.05), and percent medial thickness of the PA (12.9 +/- 0.3% versus 21.4 +/- 0.4%, P < 0.01) compared with controls. IL-10 significantly reduced macrophage infiltration and vascular cell proliferation in the remodeled PA in vivo. It also significantly decreased the lung levels of transforming growth factor-beta(1) and IL-6, which are indicative of PA remodeling. In addition, IL-10 increased the lung level of heme oxygenase-1, which strongly prevents PA remodeling. In vitro analysis revealed that IL-10 significantly inhibited excessive proliferation of cultured human PA smooth muscle cells treated with transforming growth factor-beta(1) or the heme oxygenase inhibitor tin protoporphyrin IX. Thus, IL-10 prevented the development of monocrotaline-induced PAH, and these results provide new insights into the molecular mechanisms of human PAH. - Kiwamu Akagi, Ryosuke Uchibori, Kensei Yamaguchi, Keiko Kurosawa, Yoichiro Tanaka, Tomoko KozuBIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 352 3 728 - 732 2007年01月 [査読有り][通常論文]
Activating mutations of RAS are frequently observed in subsets of human cancers, indicating that RAS activation is involved in tumorigenesis. Here, we identified and characterized a novel G to T transversion mutation of the K-ras gene at the third position of codon 19 (TTG) which substituted phenylalanine for leucine in 3 primary colon carcinomas. Biological and biochemical activity was examined using transformed NIH3T3 cells expressing mutant or wild-type K-ras. Transformants harboring the K-ras mutation at codon 19 showed proliferative capacity under serum-starved conditions, less contact inhibition, anchorage-independent growth, tumorigenicity in nude mice and elevation of active Ras-GTP levels. These results indicated that this novel mutation possesses high oncogenic activity. (c) 2006 Elsevier Inc. All rights reserved. - T Okada, R Uchibori, M Iwata-Okada, M Takahashi, T Nomoto, M Nonaka-Sarukawa, T Ito, Y Liu, H Mizukami, A Kume, E Kobayashi, K OzawaMOLECULAR THERAPY 13 4 738 - 746 2006年04月 [査読有り][通常論文]
The transduction of cancer cells using recombinant adeno-associated virus (rAAV) occurs with low efficiency, which limits its utility in cancer gene therapy. We have previously sought to enhance rAAV-mediated transduction of cancer cells by applying DNA-damaging stresses. In this study, we examined the effects of the histone deacetylase inhibitor FR901228 on tumor transduction mediated by rAAV types 2 and 5. FR901228 treatment significantly improved the expression of the transgene in four cancer cell lines. The cell surface levels of alpha v integrin, FGF-R1, and PDGF-R were modestly enhanced by the presence of FR901228. These results suggest that the superior transduction induced by the HDAC inhibitor was due to an enhancement of transgene expression rather than increased viral entry. Furthermore, we characterized the association of the acetylated histone H3 in the episomal AAV vector genome by using the chromatin immunoprecipitation assay. The results suggest that the superior transduction may be related to the proposed histone-associated chromatin form of the rAAV concatemer in transduced cells. In the analysis with subcutaneous tumor models, strong enhancement of the transgene expression as well as therapeutic effect was confirmed in vivo. The use of this HDAC inhibitor may enhance the utility of rAAV-mediated transduction strategies for cancer gene therapy. - T Okada, T Nomoto, T Yoshioka, M Nonaka-Sarukawa, T Ito, T Ogura, M Iwata-Okada, R Uchibori, K Shimazaki, H Mizukami, A Kume, K OzawaHUMAN GENE THERAPY 16 10 1212 - 1218 2005年10月 [査読有り][通常論文]
Adenovirus and adeno-associated virus (AAV) vectors are increasingly used for gene transduction experiments. However, to produce a sufficient amount of these vectors for in vivo experiments requires large-capacity tissue culture facilities, which may not be practical in limited laboratory space. We describe here a large-scale method to produce adenovirus and AAV vectors with an active gassing system that uses large culture vessels to process labor-and cost-effective infection or transfection in a closed system. Development of this system was based on the infection or transfection of 293 cells on a large scale, using a large culture vessel with a surface area of 6320 cm(2). A minipump was connected to the gas inlet of the large vessel, which was placed inside the incubator, so that the incubator atmosphere was circulated through the vessel. When active gassing was employed, the productivity of the adenovirus and AAV vectors significantly increased. This vector production system was achieved by improved CO2 and air exchange and maintenance of pH in the culture medium. Viral production with active gassing is particularly promising, as it can be used with existing incubators and the large culture vessel can readily be converted for use with the active gassing system.