Faculty Profiles - FUJIWARA Toshiyoshi

MISC - FUJIWARA Toshiyoshi

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In vivo internal tumor illumination by telomerase-dependent adenoviral GFP for precise surgical navigation

Hiroyuki Kishimoto, Ming Zhao, Katsuhiro Hayashi, Yasuo Urata, Noriaki Tanaka, Toshiyoshi Fujiwara, Sheldon Penman, Robert M. Hoffman

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 106 ( 34 ) 14514 - 14517 2009.8

Language：English Publisher：NATL ACAD SCIENCES

Cancer surgery requires the complete and precise identification of malignant tissue margins including the smallest disseminated lesions. Internal green fluorescent protein (GFP) fluorescence can intensely illuminate even single cells but requires GFP sequence transcription within the cell. Introducing and selectively activating the GFP gene in malignant tissue in vivo is made possible by the development of OBP-401, a telomerase-dependent, replication-competent adenovirus expressing GFP. This potentially powerful adjunct to surgical navigation was demonstrated in 2 nude mouse models that represent difficult surgical challenges-the resection of widely disseminated cancer. HCT-116, a model of intraperitoneal disseminated human colon cancer, was labeled by virus injection into the peritoneal cavity. A549, a model of pleural dissemination of human lung cancer, was labeled by virus administered into the pleural cavity. Only the malignant tissue fluoresced brightly in both models. In the intraperitoneal model of disseminated cancer, fluorescence-guided surgery enabled resection of all tumor nodules labeled with GFP by OBP-401. The data in this report suggest that adenoviral-GFP labeling tumors in patients can enable fluorescence-guided surgical navigation.

DOI： 10.1073/pnas.0906388106

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Diagnostic potential and limitation of imaging cancer cells in cytological samples using telomerase-specific replicative adenovirus

Yoshiko Maida, Satoru Kyo, Junko Sakaguchi, Yasunari Mizumoto, Manabu Hashimoto, Noriko Mori, Tomomi Ikoma, Mitsuhiro Nakamura, Masahiro Takakura, Yasuo Urata, Toshiyoshi Fujiwara, Masaki Inoue

INTERNATIONAL JOURNAL OF ONCOLOGY 34 ( 6 ) 1549 - 1556 2009.6

Language：English Publisher：SPANDIDOS PUBL LTD

Cytological cancer screening that targets genetic or epigenetic abnormalities may be a viable alternative to morphological screening. Detecting cancer cells by specific genetic markers helps their easy detection in cytological samples. We recently established the telomerase-specific replication-selective adenovirus OBP-401, in which the human telomerase reverse transcriptase (hTERT) gene promoter has been inserted upstream of the El genes, and in which the green fluorescent protein (GFP) gene is driven by the CMV promoter. This virus selectively replicates only in telomerase-positive cells, expressing GFP, and therefore may be a toot for cancer screening. In the present study, we first confirmed that cytological samples can easily be infected with OBP-401, allowing visualization of GFP-positive cells under fluorescent microscopy 24 h after infection. After 32 cytological samples from patients with cervical, endometrial or ovarian cancers were infected with OBP-401, GFP signals were detected in 31 (96%) of the samples. However, some normal endometrial scrapings exhibited GFP-signals, possibly due to endometrial glandular cells with constitutive telomerase activity. The ability of OBP-401 to enrich cancer cells was then tested. Cytological samples containing cervical or endometrial cancer cells were infected with OBP-401, and GFP-positive cells were sorted by flow cytometry; DNA was extracted from the GFP-positive cells. Direct DNA sequencing or methylation-specific PCR identified cancer-derived mutations or hypermethylations of tumor suppressor genes more efficiently than analyses using crude cytological samples. Thus, OBP-401-Z, based sorting of GFP-positive cells successfully enriched cancer cells, allowing efficient detection of genetic or epigenetic abnormalities in cytological samples.

DOI： 10.3892/ijo_00000284

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Antiviral activity of cidofovir against telomerase-specific replication-selective oncolytic adenovirus, OBP-301 (Telomelysin)

Masaaki Ouchi, Hitoshi Kawamura, Yasuo Urata, Toshiyoshi Fujiwara

INVESTIGATIONAL NEW DRUGS 27 ( 3 ) 241 - 245 2009.6

Language：English Publisher：SPRINGER

We constructed a replication-competent oncolytic adenovirus, OBP-301 (Telomelysin), in which human telomerase reverse transcriptase (hTERT) promoter drives E1 genes. OBP-301 is currently being used in a phase-I clinical trial for various types of tumors. Under such conditions, anti-adenoviral agents should be available for safety use against OBP-301 since any adenoviral viremia could cause severe adverse effects. Cidofovir (CDV) is an acyclic nucleoside phosphonate that has a broad antiviral activity against DNA viruses. Here, we examined the antiviral effects of CDV against OBP-301. The in vitro cytopathic effects of OBP-301 were suppressed by CDV. Moreover, CDV decreased the adenoviral E1A gene copy number after OBP-301 infection. These results suggest that CDV is a potentially useful antiviral agent for OBP-301.

DOI： 10.1007/s10637-008-9169-5

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Preclinical study of telomerase-selective oncolytic adenovirus (OBP-301) in combination with chemotherapeutic agents

Hiroshi Tazawa, Yuuri Hashimoto, Shinji Kuroda, Yasuo Urata, Toshiyoshi Fujiwara

CANCER RESEARCH 69 2009.5

Preclinical evaluation of telomerase-specific virotheranostics for human head and neck cancer

Yuji Kurihara, Toru Kojima, Tatsuo Shirota, Masashi Hatori, Yasuo Urata, Toshiyoshi Fujiwara, Satoru Shintani

CANCER RESEARCH 69 2009.5

Preclinical evaluation of synergistic effect of telomerase-specific oncolytic virotherapy and gemcitabine for human lung cancer

Dong Liu, Toru Kojima, Masaaki Ouchi, Shinji Kuroda, Yuichi Watanabe, Yuuri Hashimoto, Hideki Onimatsu, Yasuo Urata, Toshiyoshi Fujiwara

MOLECULAR CANCER THERAPEUTICS 8 ( 4 ) 980 - 987 2009.4

Language：English Publisher：AMER ASSOC CANCER RESEARCH

A phase I dose-escalation study of telomerase-specific oncolytic adenovirus, OBP-301 (Telomelysin), is now under way in the United States to assess feasibility and to characterize its pharmacokinetics in patients with advanced solid tumors. The present preclinical study investigates whether OBP-301 and a chemotherapeutic agent that is commonly used for lung cancer treatment, gemcitabine, are able to enhance antitumor effects in vitro and in vivo. The antitumor effects of OBP-301 infection and gemcitabine were evaluated by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay. In vivo antitumor effects of intratumoral injection of OBP-301 in combination with systemic administration of gemcitabine were assessed on nu/nu mice s.c. xenografted with human lung tumors. OBP-301 infection combined with gemcitabine resulted in very potent synergistic cytotoxicity in human lung cancer cells. The three human lung cancer cell lines treated with OBP-301 for 24 hours tended to accumulate in S phase compared with controls. The proportion of cells in S phase increased from 43.85% to 56.41% in H460 cells, from 46.72% to 67.09% in H322 cells, and from 38.22% to 57.67% in H358 cells. Intratumoral injection of OBP-301 combined with systemic administration of gemcitabine showed therapeutic synergism in human lung tumor xenografts. Our data suggest that the combination of OBP-301 and gemcitabine enhances the antitumor effects against human lung cancer. We also found that the synergistic mechanism may be due to OBP-301-mediated cell cycle accumulation in S phase. These results have important implications for the treatment of human lung cancer. [Mol Cancer Ther 2009;8(4):980-7]

DOI： 10.1158/1535-7163.MCT-08-0901

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Telomerase-Specific Virotheranostics for Human Head and Neck Cancer

Yuji Kurihara, Yuichi Watanabe, Hideki Onimatsu, Toru Kojima, Tatsuo Shirota, Masashi Hatori, Dong Liu, Satoru Kyo, Hiroyuki Mizuguchi, Yasuo Urata, Satoru Shintani, Toshiyoshi Fujiwara

CLINICAL CANCER RESEARCH 15 ( 7 ) 2335 - 2343 2009.4

Language：English Publisher：AMER ASSOC CANCER RESEARCH

Purpose: Long-term outcomes of patients with squamous cell carcinoma of the head and neck (SCCHN) remain unsatisfactory despite advances in combination of treatment modalities. SCCHN is characterized by locoregional spread and it is clinically accessible, making it an attractive target for intratumoral biological therapies.
Experimental Design: OBP-301 is a type 5 adenovirus that contains the replication cassette in which the human telomerase reverse transcriptase promoter drives expression of the E1 genes. OBP-401 contained the replication cassette and the green fluorescent protein (GFP) gene. The antitumor effects of OBP-301 were evaluated in vitro by the sodium 30-[1-(phenylaminocarbonyl) -3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate assay and in vivo in an orthotopic xenograft model. Virus spread into the lymphatics was also orthotopically assessed by using OBP-401.
Results: Intratumoral injection of OBP-301 resulted in the shrinkage of human SCCHN tumors orthotopically implanted into the tongues of BALB/c nu/nu mice and significantly recovered weight loss by enabling oral ingestion. The levels of GFP expression following ex vivo infection of OBP-401 may be of value as a positive predictive marker for the outcome of telomerase-specific virotherapy. Moreover, whole-body fluorescent imaging revealed that intratumorally injected OBP-401 Could visualize the metastatic lymph nodes, indicating the ability of the virus to traffic to the regional lymphatic area and to selectively replicate in neoplastic lesions, resulting in GFP expression and cell death in metastatic lymph nodes.
Conclusions: These results illustrate the potential of telomerase-specific oncolytic viruses for a novel therapeutic and diagnostic approach, termed theranostics, for human SCCHN.

DOI： 10.1158/1078-0432.CCR-08-2690

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SY-1-3 テロメラーゼ活性を標的とするウイルス製剤OBP-301の臨床応用 : 固形腫瘍に対する第1相臨床試験(外科領域における遺伝子治療・再生医療の問題点と今後の展開,シンポジウム,第109回日本外科学会定期学術集会)

香川俊輔, 宇野太, 橋本悠里, 浦田泰生, 田中紀章, 藤原俊義

日本外科学会雑誌 110 ( 2 ) 82 - 82 2009.2

Telomerase-specific virotherapy in an animal model of human head and neck cancer

Oumi Nakajima, Atsuko Matsunaga, Daiju Ichimaru, Yasuo Urata, Toshiyoshi Fujiwara, Koji Kawakami

MOLECULAR CANCER THERAPEUTICS 8 ( 1 ) 171 - 177 2009.1

Language：English Publisher：AMER ASSOC CANCER RESEARCH

Telomerase-specific replication-competent adenovirus, Telomelysin (OBP-301), has a human telomerase reverse transcriptase promoter that regulates viral replication and efficiently kills human cancer cells. The objectives of this study are to examine the effects of OBP-301 in squamous cell carcinoma of the head and neck cells in vitro and in xenografted animals in vivo. OBP-301 was found to be cytotoxic to the YCUT892, KCCT873, KCCT891, KCCL871, YCUM862, HN12, and KCCOR891 cell lines in vitro. However, the level of cytotoxicity is not correlated with the expression levels of adenoviral receptors, which may be required for adenoviral infection in squamous cell carcinoma of the head and neck cells. OBP-301 shows remarkable antitumor activity against established s.c. KCCT873 tumors in immunodeficient animals in a dose-dependent manner. In addition, no significant toxicity was observed in animals receiving treatment. These results suggest that OBP-301 is a novel therapeutic agent with promise for the treatment of human head and neck cancers. [Mol Cancer Ther 2009(1): 171 - 7]

DOI： 10.1158/1535-7163.MCT-08-0620

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Gene therapy and virotherapy for human cancer

Japanese journal of clinical medicine 67 306 - 313 2009.1

A novel antiangiogenic effect for telomerase-specific virotherapy through host immune system

Ikeda Y, Kojima T, Kuroda S, Endo Y, Sakai R, Hioki M, Kishimoto H, Uno F, Kagawa S, Watanabe Y, Hashimoto Y, Urata Y, Tanaka N, Fujiwara T

J Immunol 182 1763 - 1769 2009

RFA前のPET/CTにおけるSUVとRFA後の腫瘍再発との関連性について

原田聡介, 奥村能啓, 郷原英夫, 平木隆夫, 加藤勝也, 佐藤修平, 三村秀文, 丸中三菜子, 佐野由文, 藤原俊義, 加地充昌, 金澤右

肺癌 49 ( 5 ) 2009

肺癌RFA3,6カ月後のPET/CTの集積程度の検討

奥村能啓, 郷原英夫, 井上大作, 平木隆夫, 三村秀文, 金澤右, 佐野由文, 藤原俊義, 加地充昌

IVR 24 ( 4 ) 2009

Autophagy-inducing agents augment the antitumor effect of telomerase-selective oncolytic adenovirus OBP-405 on glioblastoma cells

T. Yokoyama, E. Iwado, Y. Kondo, H. Aoki, Y. Hayashi, M. M. Georgescu, R. Sawaya, K. R. Hess, G. B. Mills, H. Kawamura, Y. Hashimoto, Y. Urata, T. Fujiwara, S. Kondo

GENE THERAPY 15 ( 17 ) 1233 - 1239 2008.9

Language：English Publisher：NATURE PUBLISHING GROUP

Oncolytic adenoviruses are a promising tool in cancer therapy. In this study, we characterized the role of autophagy in oncolytic adenovirus-induced therapeutic effects. OBP-405, an oncolytic adenovirus regulated by the human telomerase reverse transcriptase promoter (hTERT-Ad, OBP-301) with a tropism modification (RGD) exhibited a strong antitumor effect on glioblastoma cells. When autophagy was inhibited pharmacologically, the cytotoxicity of OBP-405 was attenuated. In addition, autophagy-deficient Atg5 mouse embryonic fibroblasts (MEFs) were less sensitive than wild-type MEFs to OBP-405. These findings indicate that OBP-405-induced autophagy is a cell killing effect. Moreover, autophagy-inducing therapies (temozolomide and rapamycin) synergistically sensitized tumor cells to OBP-405 by stimulating the autophagic pathway without altering OBP-405 replication. Mice harboring intracranial tumors treated with OBP-405 and temozolomide survived significantly longer than those treated with temozolomide alone, and mice treated with OBP-405 and the rapamycin analog RAD001 survived significantly longer than those treated with RAD001 alone. The observation that autophagy inducers increase OBP-405 antitumor activity suggests a novel strategy for treating patients with glioblastoma.

DOI： 10.1038/gt.2008.98

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Understanding and exploiting hTERT promoter regulation for diagnosis and treatment of human cancers

Satoru Kyo, Masahiro Takakura, Toshiyoshi Fujiwara, Masaki Inoue

CANCER SCIENCE 99 ( 8 ) 1528 - 1538 2008.8

Language：English Publishing type：Book review, literature introduction, etc. Publisher：WILEY-BLACKWELL

Telomerase activation is a critical step for human carcinogenesis through the maintenance of telomeres, but the activation mechanism during carcinogenesis remains unclear. Transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene is the major mechanism for cancer-specific activation of telomerase, and a number of factors have been identified to directly or indirectly regulate the hTERT promoter, including cellular transcriptional activators (c-Myc, Sp1, HIF-1, AP2, ER, Ets, etc.) as well as the repressors, most of which comprise tumor suppressor gene products, such as p53, WT1, and Menin. Nevertheless, none of them can clearly account for the cancer specificity of hTERT expression. The chromatin structure via the DNA methylation or modulation of nucleosome histones has recently been suggested to be important for regulation of the hTERT promoter. DNA unmethylation or histone methylation around the transcription start site of the hTERT promoter triggers the recruitment of histone acetyltransferase (HAT) activity, allowing hTERT transcription. These facts prompted us to apply these regulatory mechanisms to cancer diagnostics and therapeutics. Telomerase-specific replicative adenovirus (Telomelysin, OBP-301), in which E1A and E1B genes are driven by the hTERT promoter, has been developed as an oncolytic virus that replicates specifically in cancer cells and causes cell death via viral toxicity. Direct administration of Telomelysin was proved to effectively eradicate solid tumors in vivo, without apparent adverse effects. Clinical trials using Telomelysin for cancer patients with progressive stages are currently ongoing. Furthermore, we incorporated green fluorescent protein gene (GFP) into Telomelysin (TelomeScan, OBP-401). Administration of TelomeScan into the primary tumor enabled the visualization of cancer cells under the cooled charged-coupled device (CCD) camera, not only in primary tumors but also the metastatic foci. This technology can be applied to intraoperative imaging of metastatic lymphnodes. Thus, we found novel tools for cancer diagnostics and therapeutics by utilizing the hTERT promoter. (Cancer Sci 2008; 99: 1528-1538)

DOI： 10.1111/j.1349-7006.2008.00878.x

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Combination of oncolytic adenovirotherapy and Bax gene therapy in human cancer xenografted models. Potential merits and hurdles for combination therapy

Masayoshi Hioki, Shunsuke Kagawa, Toshiya Fujiwara, Ryo Sakai, Turn Kojima, Yuichi Watanabe, Yuuri Hashimoto, Futoshi Uno, Norialki Tanaka, Toshiyoshi Fujiwara

INTERNATIONAL JOURNAL OF CANCER 122 ( 11 ) 2628 - 2633 2008.6

Language：English Publisher：WILEY-LISS

Cancer gene therapy and oncolytic virotherapy have been studied extensively. However, their clinical application is hampered by their weak anticancer activity. We previously constructed a replicating adenovirus (OBP-301, Telomelysin), in which the human telomerase reverse transcriptase (hTERT) promoter drives expression of the adenoviral El genes, and causes selective lysis of human cancer cells. We hypothesized that combination adenoviral therapy containing OBP-301 and a nonreplicating adenovirus expressing the proapoptotic Bax gene could overcome the weakness and augment the anticancer efficacy of each modality. Combination treatment resulted in marked Bax protein expression and enhanced efficacy in in vitro cell viability assay, when compared with either single treatment. However, combination treatment was not as effective in suppressing both subcutaneous and pleural disseminated tumors compared with OBP-301 treatment alone. Further investigation revealed that combination treatment resulted in suppressed EIA protein expression associated with reduced viral replication. Our results suggest that Bax gene therapy in combination with oncolytic adenovirotherapy potentially augments their antitumor activity, but further improvements may be required to maximize the combinatorial effect in vivo, for the Bax gene expression to avoid interference with production of the oncolytic virus. (c) 2008 Wiley-Liss, Inc.

DOI： 10.1002/ijc.23438

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Direct and distant antitumor effects of a telomerase-selective oncolytic adenoviral agent, OBP-301, in a mouse prostate cancer model

P. Huang, M. Watanabe, H. Kaku, Y. Kashiwakura, J. Chen, T. Saika, Y. Nasu, T. Fujiwara, Y. Urata, H. Kumon

CANCER GENE THERAPY 15 ( 5 ) 315 - 322 2008.5

Language：English Publisher：NATURE PUBLISHING GROUP

We previously constructed OBP-301 ( Telomelysin, a telomerase-specific replication-competent adenovirus with human telomerase reverse transcriptase ( hTERT) promoter), which showed a strong anticancer effect by inducing cell lysis of human non-small cell lung cancer and colorectal cancer cells. To investigate the utility of OBP-301 for prostate cancer treatment, we herein evaluate the cell killing and antitumor effects. First, in vitro hTERT-specific adenovirus transduction in human prostate cancer cells ( LNCaP, PC3, DU145) was confirmed using OBP-401 ( Telomelysin-green fluorescent protein ( GFP)). There was no detectable GFP transduction in the human prostate normal cells ( PrEC, PrSC). Consistently, the cell-killing effect of OBP-301 was observed only in the cancer cells. Second, using an in vivo subcutaneous LNCaP tumor model in nude mice, we demonstrated that three intratumoral OBP-301 injections ( 10(7) PFU per tumor x 3 days) were sufficient to eradicate the detectable LNCaP prostate tumor. We also demonstrated that the ispilateral treatment with OBP-301 significantly suppressed contralateral LNCaP tumor growth in both sides of the tumor model. Histological and immunohistochemical analyses revealed diffuse oncolytic degeneration and adenoviral E1A protein expression in both sides of the tumors. Therefore, in situ OBP-301 administration could be a promising therapeutic strategy against prostate cancer and its metastatic lesions.

DOI： 10.1038/cgt.2008.3

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DP-141-5 当科における食道癌術後深部静脈血栓症の発症要因とリスク解析(第108回日本外科学会定期学術集会)

山辻知樹, 猶本良夫, 田邊俊介, 藤原康弘, 渡辺信之, 櫻間一史, 伊藤充矢, 西川敏雄, 元木崇之, 田渕陽子, 高岡宗徳, 白川靖博, 小林直哉, 藤原俊義, 松原長秀, 平松聡, 羽井佐実, 松岡順治, 田中紀章

日本外科学会雑誌 109 ( 2 ) 626 - 626 2008.4

WS-4-1 テロメラーゼ活性を標的とした新規ウイルス製剤Telomelysin/TelomeScanの癌診断・治療への応用(第108回日本外科学会定期学術集会)

藤原俊義, 香川俊輔, 宇野太, 浦田泰生, 田中紀章

日本外科学会雑誌 109 ( 2 ) 146 - 146 2008.4

DP-086-2 食道癌術前診断におけるPET/CTの有用性についての検討(第108回日本外科学会定期学術集会)

田辺俊介, 猶本良夫, 藤原康宏, 櫻間一史, 野間和広, 白川靖博, 山辻知樹, 小林直哉, 藤原俊義, 松原長秀, 羽井佐実, 松岡順治, 田中紀章

日本外科学会雑誌 109 ( 2 ) 516 - 516 2008.4

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