MISC - SAKAGUCHI Masakiyo

The role of S100A8/A9 in acute exacerbation of pulmonary fibrosis

中村尚季, 肥後寿夫, 妹尾賢, 尾関太一, 角南良太, 山元修成, 板野純子, 谷口暁彦, 田中彩加, 木下理恵, 阪口政清, 前田嘉信, 宮原信明, 宮原信明

日本呼吸器学会誌(Web)   13   2024

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Development of inhibitors of axonal degeneration-inducing molecule SARM1 using human iPS cell-derived neurons and animal models

村田等, 安藤隆幸, 安井優, 越智俊樹, 友信奈保子, 山本健一, 木下理恵, 阪口政清

組織培養研究(Web)   41 ( 2 )   2023

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A novel function of REIC protein on tumor suppression through the downregulation of PD-L1 on cancer cells

合原勇馬, 友信奈保子, 木下理恵, 村田等, 山本健一, 難波正義, 許南浩, 公文裕己, 阪口政清

組織培養研究(Web)   41 ( 2 )   2023

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The Zn²⁺-bound form of ZEB1 plays a crucial part in the promotion of invasiveness in breast cancer cells

山本健一, 平林大輔, 丸山顕嘉, 友信奈保子, 木下理恵, KOMALASARI Ni Luh Gade Yoni, 村田等, 合原勇馬, 江帆, 山内明, 栗林太, 豊岡伸一, 井上裕介, 阪口政清

組織培養研究(Web)   41 ( 2 )   2023

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Nuclear PD-L1 facilitates disseminative activity by suppressing Protein X in triple-negative breast cancer cells.

合原勇馬, 友信奈保子, 木下理恵, 山本建一, 村田等, 阪口政清

日本癌学会学術総会抄録集(Web)   82nd   2023

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S100A8/A9 promotes bladder cancer progression by the TIRAP-TPL2 signaling upon the binding with TLR4

友信奈保子, 木下理恵, 合原勇馬, KOMALASARI Yoni, 二見淳一郎, 山内明, 近藤英作, 豊岡伸一, 阪口政清

日本癌学会学術総会抄録集(Web)   82nd   2023

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Discovery of the mechanism of breast cancer progression focusing on cell-surface annexin A2/S100A11 binding

高橋徹多, 友信奈保子, KOMALASARI Ni Luh Gede Yoni, 合原勇馬, 山本健一, 木下理恵, 村田等, 阪口政清

組織培養研究(Web)   41 ( 2 )   2023

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HRG suppresses the S100A8/A9-mediated metastasis of melanoma cells

友信奈保子, 木下理恵, 合原勇馬, KOMALASARI Ni Luh Gede Yoni, JIANG Fan, 村田等, 山本健一, 山内明, 近藤英作, 豊岡伸一, 西堀正洋, 阪口政清

組織培養研究(Web)   41 ( 2 )   2023

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In vitro evaluation of bioactivities of the intestine-inhabitant actinomycetes that are isolated from a therapeutically beneficial fecal microbiota

阪口義彦, 武晃, 後藤和義, 竹原正也, 友信奈保子, 山本健一, 菊池雄太, 坂本光央, 加藤はる, 大宮直木, 阪口政清, 永浜政博

組織培養研究(Web)   41 ( 2 )   2023

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Anti-S100A8/A9 Neutralizing Monoclonal Antibody Ameliorates Lung Injury Induced by Lung Ischemia Reperfusion Injury

K. Nakata, M. Okazaki, K. Miyoshi, S. Sugimoto, M. Sakaguchi, S. Toyooka

The Journal of Heart and Lung Transplantation   41 ( 4 )   2022.4

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DOI： 10.1016/j.healun.2022.01.040

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Inhibiting S100A8/A9 Attenuates Airway Obstruction in a Mouse Heterotopic Tracheal Transplantation Model

D. Shimizu, M. Okazaki, S. Sugimoto, R. Kinoshita, S. Kawana, Y. Kubo, K. Matsubara, K. Nakata, A. Matsukawa, M. Sakaguchi, S. Toyooka

The Journal of Heart and Lung Transplantation   41 ( 4 )   2022.4

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DOI： 10.1016/j.healun.2022.01.191

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軸索変性誘導分子SARM1の阻害剤開発

村田等, 安藤隆幸, 大磯和真, 友信奈保子, 山本健一, 木下理恵, 阪口政清

日本神経化学会大会抄録集(Web)   65th   2022

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REIC protein plays critical role in an anti-cancer immune regulation

合原勇馬, 友信奈保子, 木下理恵, 山本健一, 阪口政清

日本癌学会学術総会抄録集(Web)   81st   2022

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HRG suppresses the S100A8/A9-mediated brain tropic metastasis of melanoma cells

友信奈保子, KOMALASARI Yoni, 合原勇馬, 木下理恵, 山本健一, 山内明, 近藤英作, 豊岡伸一, 阪口政清

日本癌学会学術総会抄録集(Web)   81st   2022

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Binding of REIC/Dkk-3 to its receptor suppresses PD-L1 expression in triple negative breast cancer

吉澤智香子, 合原勇馬, 友信奈保子, 木下理恵, 二見淳一郎, 村田等, 山本健一, 阪口政清

日本分子生物学会年会プログラム・要旨集(Web)   45th   2022

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HSP90 inhibition suppresses not only cell proliferation but also chemotaxis in esophageal squamous cell carcinoma

Masahiro Yamamura, Akira Yamauchi, Naoki Katase, Shuichiro Okamoto, Masakiyo Sakaguchi, Yoshiyuki Yamaguchi

CANCER SCIENCE   112   477 - 477   2021.2

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NOX4 on lymphatic endothelial cells (LEC) plays an important role on the migration of pancreatic cancer cells.

Akira Yamauchi, Masahiro Yamamura, Naoki Katase, Nahoko Tomonobu, Rie Kinoshita, Masakiyo Sakaguchi, Shuichiro Okamoto

CANCER SCIENCE   112   347 - 347   2021.2

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キシリトールのグルタチオン制御を介したがん選択的抗がん作用の解析

友信奈保子, 合原勇馬, 木下理恵, 阪口政清

日本癌学会学術総会抄録集(Web)   80th   2021

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Eextracellular S100A11 upregulates mobility of pancreatic cancer cells through activation of surrounding fibroblasts

合原勇馬, 光井洋介, 友信奈保子, 木下理恵, 山内明, 山村真弘, 近藤英作, 豊岡伸一, 豊岡伸一, 那須保友, 阪口政清

日本癌学会学術総会抄録集(Web)   79th   2020

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Study of the xylitol-mediated selective cancer death and evaluation of the anticancer effect of xylitol in vivo

友信奈保子, 合原勇馬, 木下理恵, 阪口政清

日本癌学会学術総会抄録集(Web)   79th   2020

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S100A11は筋浸潤性膀胱癌細胞と線維芽細胞のクロストークに関与し腫瘍進行に寄与する(S100A11 contributes to tumor progression with cross talking between muscle invasive bladder cancer cells and fibroblasts)

光井 洋介, 定平 卓也, 渡部 昌実, 丸山 雄樹, 荒木 元朗, 渡邉 豊彦, 阪口 政清, 那須 保友

西日本泌尿器科   81 ( 増刊 )   138 - 138   2019.10

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Comparing effects of small molecular weight compounds on proliferation and chemotaxis of pancreatic cancer cells

Masahiro Yamamura, Akira Yamauchi, Naoki Katase, Masakiyo Sakaguchi, Yosuke Katata, Hiroaki Tanioka, Makoto Okawaki, Takeshi Nagasaka, Yoshiyuki Yamaguchi

CANCER RESEARCH   79 ( 13 )   2019.7

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2019-2188

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Melanoma cell adhesion molecule (MCAM) induces dissemination of melanoma upon S100A8/A9 binding

友信奈保子, 木下理恵, 近藤英作, 山内明, 二見淳一郎, 豊岡伸一, 阪口政清

日本癌学会学術総会抄録集(Web)   78th   2019

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S100タンパク質に着眼したがん転移機構の解明とその制御.

SAKAGUCHI Masakiyo

岡山医学会雑誌   130   135 - 139   2018.10

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分泌性S100A11-受容体RAGEシグナルに着眼した膵がん間質増大のメカニズムの解明

山本 健一, 高松 仁, 光井 洋介, 木下 理恵, 村田 等, 二見 淳一郎, 山本 靖彦, 西堀 正洋, 豊岡 伸一, 阪口 政清

日本がん免疫学会総会プログラム・抄録集   22回   117 - 117   2018.7

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膵がん進展に導く膵がん細胞 間質線維芽細胞クロストークを介在する分泌性S100A11 受容体RAGE連携の役割

光井 洋介, 山本 健一, Sumardika I Wayan, 木下 理恵, 村田 等, 二見 淳一郎, 高松 仁, 山本 靖彦, 西堀 正洋, 豊岡 伸一, 渡部 昌実, 那須 保友, 阪口 政清

日本がん免疫学会総会プログラム・抄録集   22回   156 - 156   2018.7

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Exogenous DKK-3/REIC inhibits Wnt/beta-catenin signaling and cell proliferation in human kidney cancer KPK1

Jiaqi Xu, Takuya Sadahira, Rie Kinoshita, Shun-Ai Li, Peng Huang, Koichiro Wada, Motoo Araki, Kazuhiko Ochiai, Hirofumi Noguchi, Masakiyo Sakaguchi, Yasutomo Nasu, Masami Watanabe

ONCOLOGY LETTERS   14 ( 5 )   5638 - 5642   2017.11

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The third member of the Dickkopf family (DKK-3), also known as reduced expression in immortalized cells (REIC), is a tumor suppressor present in a variety of tumor cells. Regarding the regulation of the Wnt/beta-catenin signaling pathway, exogenous DKK-1 and DKK-2 are reported to inhibit Wnt signaling by binding the associated effectors. However, whether exogenous DKK-3 inhibits Wnt signaling remains unclear. A recombinant protein of human full-length DKK-3 was used to investigate the exogenous effects of the protein in vitro in KPK1 human renal cell carcinoma cells. It was demonstrated that the expression of phosphorylated (p-)beta-catenin (inactive form as the transcriptional factor) was increased in KPK1 cells treated with the exogenous DKK-3 protein. The levels of non-p-beta-catenin (activated form of beta- catenin) were consistently decreased. It was revealed that the expression of transcription factor (TCF) 1 and c-Myc, the downstream transcription factors of the Wnt/beta-catenin signaling pathway, was inhibited following treatment with DKK-3. A cancer cell viability assay confirmed the anti-proliferative effects of exogenous DKK-3 protein, which was consistent with a suppressed Wnt/beta-catenin signaling cascade. In addition, as low-density lipoprotein receptor-related protein 6 (LRP6) is a receptor of DKK-1 and DKK-2 and their interaction on the cell surface inhibits Wnt/beta-catenin signaling, it was examined whether the exogenous DKK-3 protein affects LRP6-mediated Wnt/beta-catenin signaling. The LRP6 gene was silenced and the effects of DKK-3 on the time course of the upregulation of p-beta-catenin expression were subsequently analyzed. Notably, LRP6 depletion elevated the base level of p-beta-catenin; however, there was no significant effect on its upregulation course or expression pattern. These findings indicate that exogenous DKK-3 upregulates p-beta-catenin and inhibits Wnt/beta-catenin signaling in an LRP6-independent manner. Therefore, exogenous DKK-3 protein may inhibit the proliferation of KPK1 cells via inactivating Wnt/beta-catenin signaling.

DOI： 10.3892/ol.2017.6833

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Promising therapeutic efficacy of a novel reduced expression in immortalized cells/dickkopf-3 expressing adenoviral vector for hepatocellular carcinoma

Hiroaki Sawahara, Hidenori Shiraha, Daisuke Uchida, Hironari Kato, Ryo Kato, Atsushi Oyama, Teruya Nagahara, Masaya Iwamuro, Shigeru Horiguchi, Koichiro Tsutsumi, Mari Mandai, Tetsushige Mimura, Nozomu Wada, Yasuto Takeuchi, Kenji Kuwaki, Hideki Onishi, Shinichiro Nakamura, Masami Watanabe, Masakiyo Sakaguchi, Akinobu Takaki, Kazuhiro Nouso, Takahito Yagi, Yasutomo Nasu, Hiromi Kumon, Hiroyuki Okada

JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY   32 ( 10 )   1769 - 1777   2017.10

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Background and Aim: Reduced expression in immortalized cells (REIC)/dickkopf-3 (Dkk-3) is a tumor suppressor gene that is downregulated in various cancers. In our previous study of prostate cancer, the REIC/Dkk-3-expressing adenoviral vector (Ad-REIC) was found to induce cancer-selective apoptosis. This study recently developed a novel super gene expression (SGE) system and used this system to re-construct an Ad-REIC vector, termed the Ad-SGE-REIC, to achieve more effective therapeutic outcomes. In this study, the therapeutic effects of Ad-SGE-REIC on hepatocellular carcinoma (HCC) was assessed.
Methods: Human HCC cell lines (HLE, Huh7, HepG2, HLF, SK-Hep1, and PLC), human HCC tissues, and mouse HCC cell line (Hepa1-6) were used in this study. REIC/Dkk-3 expression was assessed by immunoblotting and immunohistochemistry. The relative cell viability and the apoptotic effect were examined in vitro, and the anti-tumor effects of Ad-SGE-REIC treatment were analyzed in the mouse xenograft model. This study additionally assessed anti-tumor immunological effects on the immunocompetent mice.
Results: REIC/Dkk-3 expression was decreased in HCC cell lines and HCC tissues. Ad-SGE-REIC reduced cell viability and induced apoptosis in HCC cell lines (HLE and Huh7), inhibited tumor growth in the mouse xenograft model, and demonstrated in vivo anti-cancer immunostimulatory effects on the HCC cell line (Hepa1-6).
Conclusions: Ad-SGE-REIC treatment not only enhanced cell killing effects in vitro but also elicited significant therapeutic effects, with tumor growth suppression, in vivo. REIC/Dkk-3 gene therapy using Ad-SGE-REIC potentially represents an innovative new therapeutic tool for HCC.

DOI： 10.1111/jgh.13757

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Identification of a novel component leading to anti-tumor activity besides the major ingredient cordycepin in Cordyceps militaris extract

Takeharu Wada, I. Wayan Sumardika, Shingo Saito, I. Made Winarsa Ruma, Eisaku Kondo, Masami Shibukawa, Masakiyo Sakaguchi

JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES   1061   209 - 219   2017.9

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In accordance with our previous study that was carried out to identify novel anti-tumor ingredients, chromatographic separation in combination with an anti-tumor activity assay was used for analysis of Cordyceps militaris extract in this study. Various modes of chromatography including reversed-phase, cation-exchange and anion exchange were used to separate components of Cordyceps militaris, which showed various chemical properties. Anti-tumor activity of each fraction was assessed by a Hoechst staining-based apoptosis assay using malignant melanoma MeWo cells. By these repeated approaches through chromatographic segregation and cell biological assay, we finally succeeded in identifying the target substance from a certain fraction that included neutral hydrophilic components using a pre-column and post-column chlorine adduct ionization LC APCI MS method. The target substance was a mono-carbohydrate, xylitol, that induced apoptotic cell death in MeWo cells but not in normal human OUMS-24 fibroblasts. This is the first study showing that Cordyceps militaris extract contains a large amount of xylitol. Thus, our results will contribute greatly to uncovering the mysterious multifunctional herbal drug Cordyceps militaris as an anti-tumor agent.

DOI： 10.1016/j.jchromb.2017.07.022

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An HNF4-microRNA-194/192 signaling axis maintains hepatic cell function

Aoi Morimoto, Mana Kannari, Yuichi Tsuchida, Shota Sasaki, Chinatsu Saito, Tsuyoshi Matsuta, Tsukasa Maeda, Megumi Akiyama, Takahiro Nakamura, Masakiyo Sakaguchi, Nobukazu Nameki, Frank J. Gonzalez, Yusuke Inoue

JOURNAL OF BIOLOGICAL CHEMISTRY   292 ( 25 )   10574 - 10585   2017.6

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Hepatocyte nuclear factor 4 (HNF4) controls the expression of liver-specific protein-coding genes. However, some microRNAs are also modulated by HNF4, and it is not known whether they are direct targets of HNF4 and whether they influence hepatic function. In this study, we found that HNF4 regulates microRNAs, indicated by marked down-regulation of miR-194 and miR-192 (miR-194/192) in liver-specific Hnf4a-null (Hnf4a(H)) mice. Transactivation of the shared miR-194/192 promoter was dependent on HNF4 expression, indicating that miR-194/192 is a target gene of HNF4. Screening of potential mRNAs targeted by miR-194/192 revealed that expression of genes involved in glucose metabolism (glycogenin 1 (Gyg1)), cell adhesion and migration (activated leukocyte cell adhesion molecule (Alcam)), tumorigenesis and tumor progression (Rap2b and epiregulin (Ereg)), protein SUMOylation (Sumo2), epigenetic regulation (Setd5 and Cullin 4B (Cln4b)), and the epithelial-mesenchymal transition (moesin (Msn)) was up-regulated in Hnf4a(H) mice. Moreover, we also found that miR-194/192 binds the 3-UTR of these mRNAs. siRNA knockdown of HNF4 suppressed miR-194/192 expression in human hepatocellular carcinoma (HCC) cells and resulted in up-regulation of their mRNA targets. Inhibition and overexpression experiments with miR-194/192 revealed that Gyg1, Setd5, Sumo2, Cln4b, and Rap2b are miR-194 targets, whereas Ereg, Alcam, and Msn are miR-192 targets. These findings reveal a novel HNF4 network controlled by miR-194/192 that may play a critical role in maintaining the hepatocyte-differentiated state by inhibiting expression of genes involved in dedifferentiation and tumorigenesis. These insights may contribute to the development of diagnostic markers for early HCC detection, and targeting of the miR-194/192 pathway could be useful for managing HCC.

DOI： 10.1074/jbc.M117.785592

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Advanced approach to prevent cancer metastasis based on S100A8/A9-mediated seed and soil theory

49 ( 7 )   359 - 362   2017.6

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Other Link： http://search.jamas.or.jp/link/ui/2017264472

Innovative approach to prevent cancer metastasis based on S100A8/A9-mediated seed and soil theory

49 ( 3 )   143 - 146   2017.3

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Other Link： http://search.jamas.or.jp/link/ui/2017181594

Distant Bystander Effect of REIC/DKK3 Gene Therapy Through Immune System Stimulation in Thoracic Malignancies

Ken Suzawa, Kazuhiko Shien, Huang Peng, Masakiyo Sakaguchi, Masami Watanabe, Shinsuke Hashida, Yuho Maki, Hiromasa Yamamoto, Shuta Tomida, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Yasutomo Nasu, Hiromi Kumon, Shinichiro Miyoshi, Shinichi Toyooka

ANTICANCER RESEARCH   37 ( 1 )   301 - 307   2017.1

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Background: Reduced expression in immortalized cell (REIC)/Dickkoph-3 (DKK3) is a tumor-suppressor gene, and its overexpression by adenovirus vector (Ad-REIC) exhibits a remarkable therapeutic effect on various human cancer types through a mechanism triggered by endoplasmic reticulum stress. Materials and Methods: We examined the direct anti-tumor effect of Ad-REIC gene therapy on lung cancer and malignant mesothelioma cell lines in vitro, and the distant bystander effect using immunocompetent mouse allograft models with bilateral flank tumors. Results: Ad-REIC treatment showed antitumor effect in many lung cancer and malignant mesothelioma cell lines in vitro. In an in vivo model, Ad-REIC treatment inhibited the growth not only of directly treated tumors but also of distant untreated tumors. By immunohistochemical analysis, infiltration of T-cells and natural killer (NK) cells and expression of the major histocompatibility complex (MHC) class I molecules were observed in bilateral tumors. Conclusion: Ad-REIC treatment not only had a direct antitumor effect but also an indirect bystander effect through stimulation of the immune system.

DOI： 10.21873/anticanres.11321

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転移先臓器を感知する受容体群の分子制御機構の解明とその応用. Advanced approach to prevent cancer metastasis based on S100A8/A9-mediated seed and soil theory Innovative approach to prevent cancer metastasis based on S100A8/A9-mediated seed and soil theory.

阪口政清, 木下理恵, 村田 等, 山本健一, 許 南浩, 日比野 利彦

月刊「細胞」6月号   49 ( 7 )   43 - 46   2017

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転移先臓器を感知する受容体群Innovative approach to prevent cancer metastasis based on S100A8/A9-mediated seed and soil theory.

阪口政清, 木下理恵, 村田 等, 山本健一, 許 南浩, 日比野 利彦

月刊「細胞」3月号   49 ( 3 )   39 - 42   2017

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S100-SPECT uncovers cellular and molecular events of pre-metastatic niche formation and following organ-specific cancer metastasis.

Sakaguchi M

Theranostics   7 ( 10 )   2649 - 2651   2017

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ß-1,3-galactosyl-O-glycosyl-glycoprotein ß-1,6-N-acetylglucosaminyltransferase 3 Increases MCAM Stability, Which Enhances S100A8/A9-Mediated Cancer Motility.

Sumardika IW, Youyi C, Kondo E, Inoue Y, Ruma IMW, Murata H, Kinoshita R, Yamamoto KI, Tomida S, Shien K, Satoh H, Yamauchi A, Futami J, Putranto EW, Hibino T, Toyooka S, Nishibori M, Sakaguchi M

Oncol Res   2017

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【アトピー性皮膚炎の病態研究】 Neuroplastin βとextracellular matrix metallo-protease inducerはS100A8/A9に対する機能的な受容体であり、ケラチノサイトの増殖とアトピーの皮膚炎症の増強に関与している

阪口 政清, 山本, 真実, 宮井 雅史, 木下 理恵, 村田 等, 山本 健一, 森実 真, 岩月 啓氏, 許 南浩, 坪井 良治, 日比野 利彦

臨床免疫・アレルギー科   67 ( 6 )   594 - 598   2017

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Critical role of novel receptors to S100A8/A9, EMMPRIN and NPTNβ, on keratinocyte growth and inflammation in atopic dermatitis

49 ( 6 )   594 - 598   2017

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Active Secretion of Dimerized S100A11 Induced by the Peroxisome in Mesothelioma Cells. International journal

Satomi Saho, Hiroki Satoh, Eisaku Kondo, Yusuke Inoue, Akira Yamauchi, Hitoshi Murata, Rie Kinoshita, Ken-Ichi Yamamoto, Junichiro Futami, Endy Widya Putranto, I Made Winarsa Ruma, I Wayan Sumardika, Chen Youyi, Ken Suzawa, Hiromasa Yamamoto, Junichi Soh, Shuta Tomida, Yoshihiko Sakaguchi, Ken Saito, Hidekazu Iioka, Nam-Ho Huh, Shinichi Toyooka, Masakiyo Sakaguchi

Cancer microenvironment : official journal of the International Cancer Microenvironment Society   9 ( 2-3 )   93 - 105   2016.12

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S100A11, a small Ca2+ binding protein, acts extracellularly as a mediator of cancer progression. That raises the question of how a protein that lacks the classical secretory signal is able to be secreted outside cells without being damaged. Some insights into this question have been obtained, and there has been accumulating evidence indicating a pivotal role of a non-classical vesicle-mediated pathway using lysosomes or peroxisomes for the protein secretion. To obtain a more precise insight into the secretory mechanism of S100A11, we first screened representative cancer cells exhibiting significantly active secretion of S100A11. From the results of profiling, we turned our attention to aggressive cancer mesothelioma cells. In mesothelioma cells, we found that abundant dimeric S100A11 was produced selectively in the peroxisome after transportation of monomeric S100A11 through an interaction with PEX14, a peroxisome membrane protein, resulting in peroxisomal secretion of dimerized S100A11. In an extracellular environment in vitro, dimerized S100A11 promoted mesothelial cell invasion indirectly with the help of fibroblast cells. Overall, the results indicate that the peroxisome functions as an essential vesicle for the production of dimerized S100A11 and the subsequent secretion of the protein from mesothelioma cells and that peroxisome-mediated secretion of dimerized S100A11 might play a critical role in mesothelioma progression in a tumor microenvironment.

DOI： 10.1007/s12307-016-0185-2

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Interaction of cytokeratin 19 head domain and HER2 in the cytoplasm leads to activation of HER2-Erk pathway

Tomoaki Ohtsuka, Masakiyo Sakaguchi, Hiromasa Yamamoto, Shuta Tomida, Katsuyoshi Takata, Kazuhiko Shien, Shinsuke Hashida, Tomoko Miyata-Takata, Mototsugu Watanabe, Ken Suzawa, Junichi Soh, Chen Youyi, Hiroki Sato, Kei Namba, Hidejiro Torigoe, Kazunori Tsukuda, Tadashi Yoshino, Shinichiro Miyoshi, Shinichi Toyooka

SCIENTIFIC REPORTS   6   39557 - 39557   2016.12

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HER2 is a receptor tyrosine kinase and its upregulation via activating mutations or amplification has been identified in some malignant tumors, including lung cancers. Because HER2 can be a therapeutic target in HER2-driven malignancies, it is important to understand the molecular mechanisms of HER2 activation. In the current study, we identified that cytokeratin 19 (KRT19) binds to HER2 at the inside face of plasma membrane. HER2 and KRT19, which were concurrently introduced to a human embryonic kidney 293 T cells, revealed an association with each other and resulted in phosphorylation of HER2 with the subsequent activation of a downstream Erk-associated pathway. A binding assay revealed that both the NH2-terminal head domain of KRT19 and the COOH-terminal domain of HER2 were essential for their binding. To investigate the impact of the interaction between HER2 and KRT19 in lung cancer, we examined their expressions and localizations in lung cancers. We found that KRT19 was highly expressed in HER2-positive lung cancer cells, and KRT19 and HER2 were co-localized at the cell membrane. In conclusion, we found that KRT19 intracellularly binds to HER2, playing a critical role in HER2 activation.

DOI： 10.1038/srep39557

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Identification of an S100A8 Receptor Neuroplastin-beta and its Heterodimer Formation with EMMPRIN

Masakiyo Sakaguchi, Mami Yamamoto, Masashi Miyai, Tatsuo Maeda, Junichiro Hiruma, Hitoshi Murata, Rie Kinoshita, I. Made Winarsa Ruma, Endy Widya Putranto, Yusuke Inoue, Shin Morizane, Nam-Ho Huh, Ryoji Tsuboi, Toshihiko Hibino

JOURNAL OF INVESTIGATIVE DERMATOLOGY   136 ( 11 )   2240 - 2250   2016.11

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We previously reported a positive feedback loop between S100A8/A9 and proinflammatory cytokines mediated by extracellular matrix metalloproteinase inducer, an S100A9 receptor. Here, we identify neuroplastin-beta as an unreported S100A8 receptor. Neuroplastin-beta and extracellular matrix metalloproteinase inducer form homodimers and a heterodimer, and they are co-localized on the surface of cultured normal human keratinocytes. Knockdown of both receptors suppressed cell proliferation and proinflammatory cytokine induction. Upon stimulation with S100A8, neuroplastin-beta recruited GRB2 and activated extracellular signal-regulated kinase, resulting in keratinocyte proliferation. Keratinocyte proliferation in response to inflammatory stimuli was accelerated in involucrin promoter-driven S100A8 transgenic mice. Further, S100A8 and S100A9 were strongly up-regulated and co-localized in lesional skin of atopic dermatitis patients. Our results indicate that neuroplastin-beta and extracellular matrix metalloproteinase inducer form a functional heterodimeric receptor for S100A8/A9 heterodimer, followed by recruitment of specific adaptor molecules GRB2 and TRAF2, and this signaling pathway is involved in activation of both keratinocyte proliferation and skin inflammation in atopic skin. Suppression of this pathway might have potential for treatment of skin diseases associated with chronic inflammation such as atopic dermatitis.

DOI： 10.1016/j.jid.2016.06.617

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RAGEはロイコトリエンB4第一受容体BLT1と機能的に相互作用する

市木 貴子, 古賀 友紹, 奥野 利明, 佐伯 和子, 阪口 政清, 山本 靖彦, 横溝 岳彦

日本生化学会大会プログラム・講演要旨集   89回   [2P - 089]   2016.9

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A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

Tetsuo Oka, Kazuhiko Kurozumi, Yosuke Shimazu, Tomotsugu Ichikawa, Joji Ishida, Yoshihiro Otani, Toshihiko Shimizu, Yusuke Tomita, Masakiyo Sakaguchi, Masami Watanabe, Yasutomo Nasu, Hiromi Kumon, Isao Date

SCIENTIFIC REPORTS   6   33319 - 33319   2016.9

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Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87 Delta EGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.

DOI： 10.1038/srep33319

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Novel REIC/Dkk-3-encoding adenoviral vector as a promising therapeutic agent for pancreatic cancer

H. Sawahara, H. Shiraha, D. Uchida, H. Kato, T. Nagahara, M. Iwamuro, J. Kataoka, S. Horiguchi, M. Watanabe, M. Sakaguchi, A. Takaki, K. Nouso, Y. Nasu, H. Kumon, H. Okada

CANCER GENE THERAPY   23 ( 8 )   278 - 283   2016.8

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Reduced expression in immortalized cells (REIC)/dickkopf-3 (Dkk-3), a tumor suppressor gene, is downregulated in various cancers. We previously reported the tumor-inhibitory effects of the REIC/Dkk-3 gene, delivered by a conventional adenoviral vector (Ad-CAG-REIC) in pancreatic cancer. Here, we developed an Ad-REIC vector with a novel gene expression system, termed the super gene expression (SGE) system, and assessed its therapeutic effects relative to those of Ad-CAG-REIC in pantreatic cancer cells. Human pancreatic cancer cell lines ASPC1 and MIAPaCa2 were used. REIC/Dkk-3 expression was assessed by western blot analysis. Relative cell viability and apoptotic effects were examined in vitro. The anti-tumor effects of Ad-REIC treatment were assessed in the mouse xenograft model. Compared with Ad-CAG-REIC, Ad-SGE-REIC elicited a significant increase in REIC protein expression in the cells studied, Relative to Ad-CAG-REIC, Ad-SGE-REIC reduced cell viability and induced apoptosis in the ASPC1 and MIAPaCa2 cell lines in vitro, and achieved superior tumor growth inhibition in the mouse xenograft model. Compared with conventional Ad-REIC agents, Ad-SGE-REIC provided enhanced inhibitory effects against tumor growth. Our results indicate that Ad-SGE-REIC is an innovative therapeutic tool for pancreatic cancer.

DOI： 10.1038/cgt.2016.31

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MCAM, as a novel receptor for S100A8/A9, mediates progression of malignant melanoma through prominent activation of NF-κB and ROS formation upon ligand binding. International journal

I Made Winarsa Ruma, Endy Widya Putranto, Eisaku Kondo, Hitoshi Murata, Masami Watanabe, Peng Huang, Rie Kinoshita, Junichiro Futami, Yusuke Inoue, Akira Yamauchi, I Wayan Sumardika, Chen Youyi, Ken-Ichi Yamamoto, Yasutomo Nasu, Masahiro Nishibori, Toshihiko Hibino, Masakiyo Sakaguchi

Clinical & experimental metastasis   33 ( 6 )   609 - 27   2016.8

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The dynamic interaction between tumor cells and their microenvironment induces a proinflammatory milieu that drives cancer development and progression. The S100A8/A9 complex has been implicated in chronic inflammation, tumor development, and progression. The cancer microenvironment contributes to the up-regulation of this protein complex in many invasive tumors, which is associated with the formation of pre-metastatic niches and poor prognosis. Changing adhesive preference of cancer cells is at the core of the metastatic process that governs the reciprocal interactions of cancer cells with the extracellular matrices and neighboring stromal cells. Cell adhesion molecules (CAMs) have been confirmed to have high-level expression in various highly invasive tumors. The expression and function of CAMs are profoundly influenced by the extracellular milieu. S100A8/A9 mediates its effects by binding to cell surface receptors, such as heparan sulfate, TLR4 and RAGE on immune and tumor cells. RAGE has recently been identified as an adhesion molecule and has considerably high identity and similarity to ALCAM and MCAM, which are frequently over-expressed on metastatic malignant melanoma cells. In this study, we demonstrated that ALCAM and MCAM also function as S100A8/A9 receptors as does RAGE and induce malignant melanoma progression by NF-κB activation and ROS formation. Notably, MCAM not only activated NF-κB more prominently than ALCAM and RAGE did but also mediated intracellular signaling for the formation of lung metastasis. MCAM is known to be involved in malignant melanoma development and progression through several mechanisms. Therefore, MCAM is a potential effective target in malignant melanoma treatment.

DOI： 10.1007/s10585-016-9801-2

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An efficient method for the preparation of preferentially heterodimerized recombinant S100A8/A9 coexpressed in Escherichia coli

Junichiro Futami, Yuki Atago, Akari Azuma, Endy Widya Putranto, Rie Kinoshita, Hitoshi Murata, Masakiyo Sakaguchi

Biochemistry and Biophysics Reports   6   94 - 100   2016.7

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It is now known that multicomponent protein assemblies strictly regulate many protein functions. The S100 protein family is known to play various physiological roles, which are associated with alternative complex formations. To prepare sufficient amounts of heterodimeric S100A8 and S100A9 proteins, we developed a method for bicistronic coexpression from a single-vector system using Escherichia coli cells as a host. The complex formation between S100A8 and S100A9 appears to be dependent on the thermodynamic stability of the protein during expression. The stable S100A8/A9 heterodimer complex spontaneously formed during coexpression, and biologically active samples were purified by cation-exchange chromatography. Semi-stable homodimers of S100A8 and S100A9 were also formed when expressed individually. These results suggest that the assembly of S100 protein complexes might be regulated by expression levels of partner proteins in vivo. Because protein assembly occurs rapidly after protein synthesis, coexpression of relevant proteins is crucial for the design of multicomponent recombinant protein expression systems.

DOI： 10.1016/j.bbrep.2016.03.009

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Histidine-Rich Glycoprotein Prevents Septic Lethality through Regulation of Immunothrombosis and Inflammation

Hidenori Wake, Shuji Mori, Keyue Liu, Yuta Morioka, Kiyoshi Teshigawara, Masakiyo Sakaguchi, Kosuke Kuroda, Yuan Gao, Hideo Takahashi, Aiji Ohtsuka, Tadashi Yoshino, Hiroshi Morimatsu, Masahiro Nishibori

EBIOMEDICINE   9   180 - 194   2016.7

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Sepsis is a major cause of death worldwide. We show that a plasma protein histidine-rich glycoprotein (HRG) was decreased significantly in septic mice with cecal ligation and puncture (CLP) and supplementary treatment of septic mice with exogenous HRG improved survival, with strong inhibition of tight attachment of neutrophils to pulmonary vasculatures, subsequent immunothrombosis, DIC state, lung inflammation, hypercytokinemia, and activation of vascular endothelial cells (VECs). In contrast, knockdown of HRG by siRNA exacerbated lethality. Purified human HRG reversibly induced morphological changes in human neutrophils in vitro; induction of spherical shape with reduced microvilli and adhesiveness to VECs. HRG maintained the passage of neutrophils through microcapillaries and abolished production of reactive oxygen species. These results suggested that the supplementary therapy with HRG may provide a novel strategy for the treatment of sepsis through suppression of excessive systemic inflammation and immunothrombosis by keeping circulating neutrophils quiescent and preventing uncontrolled activation of VECs. (C) 2016 The Authors. Published by Elsevier B.V.

DOI： 10.1016/j.ebiom.2016.06.003

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Antitumor effect of afatinib, as a human epidermal growth factor receptor 2-targeted therapy, in lung cancers harboring HER2 oncogene alterations

Ken Suzawa, Shinichi Toyooka, Masakiyo Sakaguchi, Mizuki Morita, Hiromasa Yamamoto, Shuta Tomida, Tomoaki Ohtsuka, Mototsugu Watanabe, Shinsuke Hashida, Yuho Maki, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi

CANCER SCIENCE   107 ( 1 )   45 - 52   2016.1

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Human epidermal growth factor receptor 2 (HER2) is a member of the HER family of proteins containing four receptor tyrosine kinases. It plays an important role in the pathogenesis of certain human cancers. In non-small-cell lung cancer (NSCLC), HER2 amplification or mutations have been reported. However, little is known about the benefit of HER2-targeted therapy for NSCLCs harboring HER2 alterations. In this study, we investigated the antitumor effect of afatinib, an irreversible epidermal growth factor receptor (EGFR)-HER2 dual inhibitor, in lung cancers harboring HER2 oncogene alterations, including novel HER2 mutations in the transmembrane domain, which we recently identified. Normal bronchial epithelial cells, BEAS-2B, ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, and G660D) showed constitutive autophosphorylation of HER2 and activation of downstream signaling. They were sensitive to afatinib, but insensitive to gefitinib. Furthermore, we examined the antitumor activity of afatinib and gefitinib in several NSCLC cell lines, and investigated the association between their genetic alterations and sensitivity to afatinib treatment. In HER2-altered NSCLC cells (H2170, Calu-3, and H1781), afatinib downregulated the phosphorylation of HER2 and EGFR as well as their downstream signaling, and induced an antiproliferative effect through G(1) arrest and apoptotic cell death. In contrast, HER2- or EGFR-non-dependent NSCLC cells were insensitive to afatinib. In addition, these effects were confirmed in vivo by using a xenograft mouse model of HER2-altered lung cancer cells. Our results suggest that afatinib is a therapeutic option as a HER2-targeted therapy for NSCLC harboring HER2 amplification or mutations.

DOI： 10.1111/cas.12845

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Modulation of leukotriene B4 receptor 1 signaling by receptor for advanced glycation end products (RAGE).

Ichiki T, Koga T, Okuno T, Saeki K, Yamamoto Y, Yamamoto H, Sakaguchi M, Yokomizo T

FASEB J   30 ( 5 )   1811 - 1822   2016

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Hepatocyte Nuclear Factor 4 alpha Controls Iron Metabolism and Regulates Transferrin Receptor 2 in Mouse Liver

Shunsuke Matsuo, Masayuki Ogawa, Martina U. Muckenthaler, Yumiko Mizui, Shota Sasaki, Takafumi Fujimura, Masayuki Takizawa, Nagayuki Ariga, Hiroaki Ozaki, Masakiyo Sakaguchi, Frank J. Gonzalez, Yusuke Inoue

JOURNAL OF BIOLOGICAL CHEMISTRY   290 ( 52 )   30855 - 30865   2015.12

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Iron is an essential element in biological systems, but excess iron promotes the formation of reactive oxygen species, resulting in cellular toxicity. Several iron-related genes are highly expressed in the liver, a tissue in which hepatocyte nuclear factor 4 alpha (HNF4 alpha) plays a critical role in controlling gene expression. Therefore, the role of hepatic HNF4 alpha in iron homeostasis was examined using liver-specific HNF4 alpha-null mice (Hnf4a(Delta H) mice). Hnf4a(Delta H) mice exhibit hypoferremia and a significant change in hepatic gene expression. Notably, the expression of transferrin receptor 2 (Tfr2) mRNA was markedly decreased in Hnf4a(Delta H) mice. Promoter analysis of the Tfr2 gene showed that the basal promoter was located at a GC-rich region upstream of the transcription start site, a region that can be transactivated in an HNF4 alpha-independent manner. HNF4 alpha-dependent expression of Tfr2 was mediated by a proximal promoter containing two HNF4 alpha-binding sites located between the transcription start site and the translation start site. Both the GC-rich region of the basal promoter and the HNF4 alpha-binding sites were required for maximal transactivation. Moreover, siRNA knockdown of HNF4 alpha suppressed TFR2 expression in human HCC cells. These results suggest that Tfr2 is a novel target gene for HNF4 alpha, and hepatic HNF4 alpha plays a critical role in iron homeostasis.

DOI： 10.1074/jbc.M115.694414

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TAE226, a Bis-Anilino Pyrimidine Compound, Inhibits the EGFR-Mutant Kinase Including T790M Mutant to Show Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells

Hiroki Otani, Hiromasa Yamamoto, Munenori Takaoka, Masakiyo Sakaguchi, Junichi Soh, Masaru Jida, Tsuyoshi Ueno, Takafumi Kubo, Hiroaki Asano, Kazunori Tsukuda, Katsuyuki Kiura, Shinji Hatakeyama, Eiji Kawahara, Yoshio Naomoto, Shinichiro Miyoshi, Shinichi Toyooka

PLOS ONE   10 ( 6 )   e0129838-e0129838   2015.6

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TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The antitumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation.

DOI： 10.1371/journal.pone.0129838

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The cysteine-rich core domain of REIC/Dkk-3 is critical for its effect on monocyte differentiation and tumor regression

Rie Kinoshita, Masami Watanabe, Peng Huang, Shun-Ai Li, Masakiyo Sakaguchi, Hiromi Kumon, Junichiro Futami

ONCOLOGY REPORTS   33 ( 6 )   2908 - 2914   2015.6

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Reduced expression in immortalized cells (REIC)/Dickkopf (Dkk)-3 is a tumor-suppressor gene and has been studied as a promising therapeutic gene for cancer gene therapy. Intratumoral injection of an adenovirus vector carrying the human REIC/Dkk-3 gene (Ad-REIC) elicits cancer cell-specific apoptosis and anticancer immune responses. The cytokine-like effect of secretory REIC/Dkk-3 on the induction of dendritic cell (DC)-like cell differentiation from monocytes plays a role in systemic anticancer immunity. In the present study, we generated recombinant full-length and N-terminally truncated REIC/Dkk-3 to characterize the biological activity of the protein. During the purification procedure, we identified a 17 kDa cysteine-rich stable product (C17-REIC) showing limited degradation. Further analysis showed that the C17-REIC domain was sufficient for the induction of DC-like cell differentiation from monocytes. Concomitant with the differentiation of DCs, the REIC/Dkk-3 protein induced the phosphorylation of glycogen synthase kinase 3 beta (GSK-3 beta) and signal transducers and activators of transcription (STAT) at a level comparable to that of granulocyte/macrophage colony-stimulating factor. In a mouse model of subcutaneous renal adenocarcinoma, intraperitoneal injection of full-length and C17-REIC proteins exerted anticancer effects in parallel with the activation of immunocompetent cells such as DCs and cytotoxic T lymphocytes in peripheral blood. Taken together, our results indicate that the stable cysteine-rich core region of REIC/Dkk-3 is responsible for the induction of anticancer immune responses. Because REIC/Dkk-3 is a naturally circulating serum protein, the upregulation REIC/Dkk-3 protein expression could be a promising option for cancer therapy.

DOI： 10.3892/or.2015.3885

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Antitumor effect of afatinib, as a HER2-targeted therapy, in lung cancers harboring HER2 oncogene alterations.

Suzawa K, Toyooka S, Sakaguchi M, Morita M, Yamamoto H, Tomida S, Ohtsuka T, Watanabe M, Hashida S, Maki Y, Soh J, Asano H, Tsukuda K, Miyoshi S

Cancer Sci   cas.12845-cas.12845   2015

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Potential role of tumor suppressor gene REIC/Dkk-3 as a regulator of skin tissue inflammation

Y. Ayabe, T. Tsuruda, M. Sakaguchi, K. Kataoka

MOLECULAR BIOLOGY OF THE CELL   26   2015

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CELL BIOLOGY  

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肺腺癌におけるHER2膜貫通領域の遺伝子変異と機能解析

山本寛斉, 阪口政清, 諏澤憲, 大塚智昭, 枝園和彦, 橋田真輔, 渡邉元嗣, 牧佑歩, 宗淳一, 岡田真典, 伊賀徳周, 三好健太郎, 杉本誠一郎, 山根正修, 大藤剛宏, 三好新一郎, 豊岡伸一

日本肺癌学会総会号   55th ( 5 )   362 - 362   2014.10

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J-GLOBAL

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Anti-cancer effects of REIC/Dkk-3-encoding adenoviral vector for the treatment of non-small cell lung cancer

Ken Suzawa, Shinichi Toyooka, Kazuhiko Shien, Norimitsu Tanaka, Masami Watanabe, Junichi Soh, Masakiyo Sakaguchi, Keitaro Matsuo, Hiromasa Yamamoto, Masashi Furukawa, Hiroaki Asano, Kazunori Tsukuda, Yasutomo Nasu, Nam-Ho Huh, Shinichiro Miyoshi, Hiromi Kumon

CANCER RESEARCH   74 ( 19 )   2014.10

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2014-2879

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DNAX-activating Protein 10 (DAP10) Membrane Adaptor Associates with Receptor for Advanced Glycation End Products (RAGE) and Modulates the RAGE-triggered Signaling Pathway in Human Keratinocytes

Masakiyo Sakaguchi, Hitoshi Murata, Yumi Aoyama, Toshihiko Hibino, Endy Widya Putranto, I. Made Winarsa Ruma, Yusuke Inoue, Yoshihiko Sakaguchi, Ken-ichi Yamamoto, Rie Kinoshita, Junichiro Futami, Ken Kataoka, Keiji Iwatsuki, Nam-ho Huh

JOURNAL OF BIOLOGICAL CHEMISTRY   289 ( 34 )   23389 - 23402   2014.8

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Background: RAGE receptor plays a critical role in many inflammatory disorders. Results: Functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated cell survival. Conclusion: DAP10 membrane adaptor is critically involved in RAGE-mediated survival signaling upon S100A8/A9 binding. Significance: This is the first report demonstrating that RAGE-mediated survival signaling is critically regulated by DAP10 interaction.
The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.

DOI： 10.1074/jbc.M114.573071

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Extract of Cordyceps militaris inhibits angiogenesis and suppresses tumor growth of human malignant melanoma cells

I. Made Winarsa Ruma, Endy Widya Putranto, Eisaku Kondo, Risayo Watanabe, Ken Saito, Yusuke Inoue, Ken-Ichi Yamamoto, Susumu Nakata, Masaji Kaihata, Hitoshi Murata, Masakiyo Sakaguchi

INTERNATIONAL JOURNAL OF ONCOLOGY   45 ( 1 )   209 - 218   2014.7

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Angiogenesis is essential for tumor development and metastasis. Among several angiogenic factors, vascular endothelial growth factor receptor (VEGF) is important for tumor-derived angiogenesis and commonly overexpressed in solid tumors. Thus, many antitumor strategies targeting VEGF have been developed to inhibit cancer angiogenesis, offering insights into the successful treatment of solid cancers. However, there are a number of issues such as harmful effects on normal vascularity in clinical trials. Taking this into consideration, we employed Cordyceps militaris as an antitumor approach due to its biological safety in vivo. The herbal medicinal mushroom Cordyceps militaris has been reported to show potential anticancer properties including anti-angiogenic capacity; however, its concrete properties have yet to be fully demonstrated. In this study, we aimed to elucidate the biological role of Cordyceps militaris extract in tumor cells, especially in regulating angiogenesis and tumor growth of a human malignant melanoma cell line. We demonstrated that Cordyceps militaris extract remarkably suppressed tumor growth via induction of apoptotic cell death in culture that links to the abrogation of VEGF production in melanoma cells. This was followed by mitigation of Akt1 and GSK-3 beta activation, while p38 alpha phosphorylation levels were increased. Extract treatment in mouse model xenografted with human melanoma cells resulted in a dramatic antitumor effect with downregulation of VEGF expression. The results suggest that suppression of tumor growth by Cordyceps militaris extract is, at least, mediated by its anti-angiogenicity and apoptosis induction capacities. Cordyceps militaris extract may be a potent antitumor herbal drug for solid tumors.

DOI： 10.3892/ijo.2014.2397

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S100A11 is required for efficient plasma membrane repair and survival of invasive cancer cells

Jyoti K. Jaiswal, Stine P. Lauritzen, Luana Scheffer, Masakiyo Sakaguchi, Jakob Bunkenborg, Sanford M. Simon, Tuula Kallunki, Marja Jaattela, Jesper Nylandsted

NATURE COMMUNICATIONS   5   a3795-a3795   2014.5

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Language：English   Publisher：NATURE PUBLISHING GROUP  

Cell migration and invasion require increased plasma membrane dynamics and ability to navigate through dense stroma, thereby exposing plasma membrane to tremendous physical stress. Yet, it is largely unknown how metastatic cancer cells acquire an ability to cope with such stress. Here we show that S100A11, a calcium-binding protein upregulated in a variety of metastatic cancers, is essential for efficient plasma membrane repair and survival of highly motile cancer cells. Plasma membrane injury-induced entry of calcium into the cell triggers recruitment of S100A11 and Annexin A2 to the site of injury. We show that S100A11 in a complex with Annexin A2 helps reseal the plasma membrane by facilitating polymerization of cortical F-actin and excision of the damaged part of the plasma membrane. These data reveal plasma membrane repair in general and S100A11 and Annexin A2 in particular as new targets for the therapy of metastatic cancers.

DOI： 10.1038/ncomms4795

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超高効率遺伝子発現システムを用いた次世代REIC遺伝子治療の開発

渡部 昌実, 阪口 政清, 賀来 春紀, 佐々木 克己, 高本 篤, 有吉 勇一, 杉本 盛人, 江原 伸, 渡辺 豊彦, 許 南浩, 那須 保友, 公文 裕巳

日本泌尿器科学会総会   102回   621 - 621   2014.4

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A novel gene expression system strongly enhances the anticancer effects of a REIC/Dkk-3-encoding adenoviral vector

Masami Watanabe, Masakiyo Sakaguchi, Rie Kinoshita, Haruki Kaku, Yuichi Ariyoshi, Hideo Ueki, Ryuta Tanimoto, Shin Ebara, Kazuhiko Ochiai, Junichiro Futami, Shun-Ai Li, Peng Huang, Yasutomo Nasu, Nam-Ho Huh, Hiromi Kumon

ONCOLOGY REPORTS   31 ( 3 )   1089 - 1095   2014.3

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Gene expression systems with various promoters, including the cytomegalovirus (CMV) promoter, have been developed to increase the gene expression in a variety of normal and cancer cells. In particular, in the clinical trials of cancer gene therapy, a more efficient and robust gene expression system is required to achieve sufficient therapeutic outcomes. By inserting the triple translational enhancer sequences of human telomerase reverse transcriptase (hTERT), Simian virus 40 (SV40) and CMV downstream of the sequence of the BGH polyA, we were able to develop a novel gene expression system that significantly enhances the expression of the genes of interest. We termed this novel gene expression cassette the super gene expression (SGE) system, and herein verify the utility of the SGE cassette for a replication-deficient adenoviral vector. We newly developed an adenoviral vector expressing the tumor suppressor, reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), based on the CMV promoter-driven SGE system (Ad-SGE-REIC) and compared the therapeutic utility of Ad-SGE-REIC with that of the conventional adenoviral vectors (Ad-CMV-REIC or Ad-CAG-REIC). The results demonstrated that the CMV promoter-SGE system allows for more potent gene expression, and that the Ad-SGE-REIC is superior to conventional adenoviral systems in terms of the REIC protein expression and therapeutic effects. Since the SGE cassette can be applied for the expression of various therapeutic genes using various vector systems, we believe that this novel system will become an innovative tool in the field of gene expression and gene therapy.

DOI： 10.3892/or.2013.2958

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Coxsackie and adenovirus receptor is a critical regulator for the survival and growth of oral squamous carcinoma cells

K. Saito, M. Sakaguchi, H. Iioka, M. Matsui, H. Nakanishi, N. H. Huh, E. Kondo

ONCOGENE   33 ( 10 )   1274 - 1286   2014.3

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Coxsackie and adenovirus receptor (CAR) is essential for adenovirus infection to target cells, and its constitutive expression in various cancerous and normal tissues has been reported. Recently, the biological role of CAR in human cancers of several different origins has been investigated with respect to tumor progression, metastasis and tumorigenesis. However, its biological function in tumor cells remains controversial. Here we report the critical role of CAR in growth regulation of oral squamous cell carcinomas (SCCs) in vitro and in vivo via the specific interaction with Rho-associated protein kinase (ROCK). Loss of endogenous CAR expression by knockdown using specific small interfering RNA (siRNA) against CAR facilitates growth suppression of SCC cells due to cell dissociation, followed by apoptosis. The consequent morphological reaction was reminiscent of anoikis, rather than epithelial-mesenchymal transition, and the dissociation of oral SCC cells was triggered not by lack of contact with extracellular matrix, but by loss of cell-to-cell contact caused by abnormal translocation of E-cadherin from surface membrane to cytoplasm. Immunoprecipitation assays of the CAR-transfected oral SCC cell line, HSC-2, with or without ROCK inhibitor (Y-27632) revealed that CAR directly associates with ROCK! and ROCKII, which results in inhibition of ROCK activity and contributes to maintenance of cell-to-cell adhesion for their growth and survival. Based on these findings, in vivo behavior of CAR-downregulated HSC-2 cells from siRNA knockdown was compared with that of normally CAR-expressing cells in intraperitoneally xenografted mouse models. The mice engrafted with CAR siRNA-pretreated HSC-2 cells showed poor formation of metastatic foci in contrast to those implanted with the control siRNA-pretreated cells. Thus, CAR substantially has an impact on growth and survival of oral SCC cells as a negative regulator of ROCK in vitro and in vivo.

DOI： 10.1038/onc.2013.66

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Anti-Cancer Effects of REIC/Dkk-3-encoding Adenoviral Vector for the Treatment of Non-small Cell Lung Cancer

Kazuhiko Shien, Norimitsu Tanaka, Masami Watanabe, Junichi Soh, Masakiyo Sakaguchi, Keitaro Matsuo, Hiromasa Yamamoto, Masashi Furukawa, Hiroaki Asano, Kazunori Tsukuda, Yasutomo Nasu, Nam-Ho Huh, Shinichiro Miyoshi, Hiromi Kumon, Shinichi Toyooka

PLOS ONE   9 ( 2 )   e87900-e87900   2014.2

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Objectives: REIC/Dkk-3 is down-regulated in a broad range of human cancer cells and is considered to function as a tumor suppressor. We previously reported that REIC/Dkk-3-expressing adenovirus vector (Ad-REIC) induced endoplasmic reticulum (ER) stress and cancer-specific apoptosis in human prostate cancer. In this study, we examined the therapeutic impact of Ad-REIC on non-small cell lung cancer (NSCLC).
Materials and Methods: We examined the anti-tumor effect of Ad-REIC on 25 NSCLC cell lines in vitro and A549 cells in vivo. Two of these cell lines were artificially established as EGFR-tyrosine kinase inhibitor (TKI) resistant sublines.
Results: Ad-REIC-treatment inhibited the cell viability by 40% or more in 13 (52%) of the 25 cell lines at multiplicity of infection (MOI) of 20 (20 MOI). These cell lines were regarded as being highly sensitive cells. The cell viability of a nonmalignant immortalized cell line, OUMS-24, was not inhibited at 200 MOI of Ad-REIC. The effects of Ad-REIC on EGFR-TKI resistant sublines were equivalent to those in the parental cell lines. Here, we demonstrated that Ad-REIC treatment activated c-Jun N-terminal kinase (JNK) in NSCLC cell lines, indicating the induction of ER stress with GRP78/BiP (GRP78) up-regulation and resulting in apoptosis. A single intratumoral injection of Ad-REIC significantly inhibited the tumorigenic growth of A549 cells in vivo. As predictive factors of sensitivity for Ad-REIC treatment in NSCLC, we examined the expression status of GRP78 and coxsackievirus and adenovirus receptor (CAR). We found that the combination of the GRP78 and CAR expressional statuses may be used as a predictive factor for Ad-REIC sensitivity in NSCLC cells.
Conclusion: Ad-REIC induced JNK activation and subsequent apoptosis in NSCLC cells. Our study indicated that Ad-REIC has therapeutic potential against NSCLC and that the expression statuses of GRP78 and CAR may predict a potential therapeutic benefit of Ad-REIC.

DOI： 10.1371/journal.pone.0087900

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Preclinical Evaluation of MicroRNA-34b/c Delivery for Malignant Pleural Mesothelioma

Tsuyoshi Ueno, Shinichi Toyooka, Takuya Fukazawa, Takafumi Kubo, Junichi Soh, Hiroaki Asano, Takayuki Muraoka, Norimitsu Tanaka, Yuho Maki, Kazuhiko Shien, Masashi Furukawa, Masakiyo Sakaguchi, Hiromasa Yamamoto, Kazunori Tsukuda, Shinichiro Miyoshi

ACTA MEDICA OKAYAMA   68 ( 1 )   23 - 26   2014.2

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The microRNA-34s (miR-34s) have p53 response elements in their 5'-flanking regions and demonstrate tumor-suppressive functions. In malignant pleural mesothelioma (MPM), we previously reported that expression of miR-34b and miR-34c (miR-34b/c) was frequently downregulated by methylation in MPM cell lines and primary tumors. The forced overexpression of miR-34b/c showed significant antitumor effects with the induction of apoptosis in MPM cells. In this study, we examined the in vivo antitumor effects of miR-34b/c using adenovirus vector on MPM. We subcutaneously transplanted NCI-H290, a human MPM cell line, into BALB/C mice and injected adenovirus vector expressing miR-34b/c, luciferase driven by the cytomegalovirus promoter (Ad-miR-34b/c or Ad-Luc), or PBS control into tumors over 5mm in diameter. A statistically significant growth inhibition of the tumor volume was observed in the Ad-miR-34b/c group from day 6 onward compared to the Ad-Luc group. The inhibition rate of Ad-miR-34b/c, compared to the tumor volume treated with Ad-Luc, was 58.6% on day 10 and 54.7% on day13. Our results indicate that adenovirus-mediated miR-34b/c gene therapy could be useful for the clinical treatment of MPM.

DOI： 10.18926/AMO/52140

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Novel Germline Mutation in the Transmembrane Domain of HER2 in Familial Lung Adenocarcinomas

Hiromasa Yamamoto, Koichiro Higasa, Masakiyo Sakaguchi, Kazuhiko Shien, Junichi Soh, Koichi Ichimura, Masashi Furukawa, Shinsuke Hashida, Kazunori Tsukuda, Nagio Takigawa, Keitaro Matsuo, Katsuyuki Kiura, Shinichiro Miyoshi, Fumihiko Matsuda, Shinichi Toyooka

JNCI-JOURNAL OF THE NATIONAL CANCER INSTITUTE   106 ( 1 )   djt338-djt338   2014.1

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We encountered a family of Japanese descent in which multiple members developed lung cancer. Using whole-exome sequencing, we identified a novel germline mutation in the transmembrane domain of the human epidermal growth factor receptor 2 (HER2) gene (G660D). A novel somatic mutation (V659E) was also detected in the transmembrane domain of HER2 in one of 253 sporadic lung adenocarcinomas. Because the transmembrane domain of HER2 is considered to be responsible for the dimerization and subsequent activation of the HER family and downstream signaling pathways, we performed functional analyses of these HER2 mutants. Mutant HER2 G660D and V659E proteins were more stable than wild-type protein. Both the G660D and V659E mutants activated Akt. In addition, they activated p38, which is thought to promote cell proliferation in lung adenocarcinoma. Our findings strongly suggest that mutations in the transmembrane domain of HER2 may be oncogenic, causing hereditary and sporadic lung adenocarcinomas.

DOI： 10.1093/jnci/djt338

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Draft genome sequence of Clostridium botulinum type B strain Osaka05, isolated from an infant patient with botulism in Japan

Yoshihiko Sakaguchi, Koji Hosomi, Jumpei Uchiyama, Yoshitoshi Ogura, Kaoru Umeda, Masakiyo Sakaguchi, Tomoko Kohda, Masafumi Mukamoto, Naoaki Misawa, Shigenobu Matsuzaki, Tetsuya Hayashi, Shunji Kozaki

Genome Announcements   2 ( 1 )   e01010-e01013   2014

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© 2014 Sakaguchi et al. Clostridium botulinum strain Osaka05, which has been isolated from an infant patient with botulism in Japan, is the first strain producing botulinum neurotoxin subtype B6. Here, we report the draft genome sequence of C. botulinum Osaka05.

DOI： 10.1128/genomeA.e01010-13

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Tumor Necrosis Factor-alpha Down-regulates a Tumor Suppressor Gene, REIC/Dkk-3, in Normal Skin Keratinocytes

K. Kataoka, M. Sakaguchi, H. Murata, N. Hashikawa, N. Huh

IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL   49   S43 - S43   2013.6

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：SPRINGER  

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DOCK7 is a critical regulator of the RAGE-Cdc42 signaling axis that induces formation of dendritic pseudopodia in human cancer cells

Ken-Ichi Yamamoto, Hitoshi Murata, Endy Widya Putranto, Ken Kataoka, Akira Motoyama, Toshihiko Hibino, Yusuke Inoue, Masakiyo Sakaguchi, Nam-Ho Huh

ONCOLOGY REPORTS   29 ( 3 )   1073 - 1079   2013.3

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Cellular migration is a fundamental process linked to cancer metastasis. Growing evidence indicates that the receptor for advanced glycation end products (RAGE) plays a pivotal role in this process. With regard to downstream signal transducers of RAGE, diaphanous-1 and activated small guanine nucleotide triphosphatases, Rac1 and Cdc42, have been identified. To obtain precise insight into the direct downstream signaling mechanism of RAGE, we screened for proteins interacting with the cytoplasmic domain of RAGE employing an immunoprecipitation-liquid chromatography coupled with an electrospray tandem mass spectrometry system. In the present study, we found that the cytoplasmic domain of RAGE interacted with an atypical DOCK180-related guanine nucleotide exchange factor, dedicator of cytokinesis protein 7 (DOCK7). DOCK7 bound to the RAGE cytoplasmic domain and transduced a signal to Cdc42, resulting in the formation of abundant highly branched filopodia-like protrusions, dendritic pseudopodia. Blocking of the function of DOCK7 greatly abrogated the formation of dendritic pseudopodia and suppressed cellular migration. These results indicate that DOCK7 functions as an essential and downstream regulator of RAGE-mediated cellular migration through the formation of dendritic pseudopodia.

DOI： 10.3892/or.2012.2191

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S100A9 is a novel ligand of EMMPRIN that promotes melanoma metastasis

Toshihiko Hibino, Masakiyo Sakaguchi, Shoko Miyamoto, Mami Yamamoto, Akira Motoyama, Junichi Hosoi, Tadashi Shimokata, Tomonobu Ito, Ryoji Tsuboi, Nam-Ho Huh

Cancer Research   73 ( 1 )   172 - 183   2013.1

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The calcium-binding proteins S100A8 and S100A9 can dimerize to form calprotectin, the release of which during tissue damage has been implicated in inflammation and metastasis. However, receptor(s) mediating the physiologic and pathophysiologic effects of this damage-associated "danger signal" are uncertain. In this study, searching for candidate calprotectin receptors by affinity isolation-mass spectrometry, we identified the cell surface glycoprotein EMMPRIN/BASIGIN (CD147/BSG). EMMPRIN specifically bound to S100A9 but not S100A8. Induction of cytokines and matrix metalloproteases (MMP) by S100A9 was markedly downregulated in melanoma cells by attenuation of EMMPRIN. We found that EMMPRIN signaling used the TNF receptor-associated factor TRAF2 distinct from the known S100-binding signaling pathway mediated by RAGE (AGER). S100A9 strongly promoted migration when EMMPRIN was highly expressed, independent of RAGE, whereas EMMPRIN blockade suppressed migration by S100A9. Immunohistologic analysis of melanomas revealed that EMMPRIN was expressed at both the invasive edge of lesions and the adjacent epidermis, where S100A9 was also strongly expressed. In epidermal-specific transgenic mice, tail vein-injected melanoma accumulated in skin expressing S100A9 but not S100A8. Together, our results establish EMMPRIN as a receptor for S100A9 and suggest the therapeutic use in targeting S100A9-EMMPRIN interactions. ©2012 AACR.

DOI： 10.1158/0008-5472.CAN-11-3843

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Identification of RAGE-coupled membran adaptor that modulates RAGE-triggered signaling pathway

M. Sakaguchi, H. Murata, T. Hibino, Y. Aoyama, K. Iwatsuki, N-H. Huh

MOLECULAR BIOLOGY OF THE CELL   24   2013

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CELL BIOLOGY  

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TRAF6 ubiquitinates and stabilizes PINK1 on the outer membrane of depolarized mitochondria through interaction with SARM1

H. Murata, M. Sakaguchi, K. Kataoka, N-H. Huh

MOLECULAR BIOLOGY OF THE CELL   24   2013

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CELL BIOLOGY  

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Preclinical biodistribution and safety study of reduced expression in immortalized cells/Dickkopf-3-encoding adenoviral vector for prostate cancer gene therapy

Morito Sugimoto, Masami Watanabe, Haruki Kaku, Shun-Ai Li, Hirofumi Noguchi, Hideo Ueki, Masakiyo Sakaguchi, Nam-Ho Huh, Yasutomo Nasu, Hiromi Kumon

ONCOLOGY REPORTS   28 ( 5 )   1645 - 1652   2012.11

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The biodistribution and safety of adenoviral vectors encoding the human REIC/Dkk-3 tumor suppressor gene (Ad-REIC) were examined in this preclinical study for in situ prostate cancer gene therapy. First, the in vitro apoptotic effects of Ad-REIC in normal and cancer cells derived from the prostate and liver were examined. Significant apoptotic effects were observed at 100 MOI (multiplicity of infection) in prostate cancer cells (LNCaP, PC3) and hepatoma cells (HEP3B and HEPG2); however, no effects were seen in normal cells. To analyze the safety of intraprostatic Ad-REIC administration, the biodistribution and histology after Ad-REIC injection were evaluated in various organs of normal male C57BL6 mice. In a supporting study, vector dissemination following intravenous injection of Ad-REIC into tail veins was determined. To evaluate whether Ad-REIC was present in the collected tissue specimens, human REIC gene detection was performed using DNA-PCR. Intraprostatic treatment administered at lower doses showed vector biodistribution into the colon, urinary bladder and prostate. At higher doses, vector dissemination was observed in tissues more distant from the prostate, including the lung, thymus, heart, liver and adrenal gland. After intravenous injection a: Ad-REIC, dissemination was observed in the liver and spleen. These results indicate that the biodistribution of Ad-REIC is determined by the dose and route of administration. Although acute inflammatory effects were observed in the prostate after intraprostatic administration at higher doses, no abnormal histological findings were noted in the other tissues, including those of intravenously treated mice. Regarding the safety of Ad-REIC administration, no deaths and no signs of toxicity or unusual behavior were observed in the mice in any treatment group. Based on these preclinical experiments, adenovirus-mediated in situ REIC/Dkk-3 gene therapy is considered to be safe for use as a treatment for human prostate cancer.

DOI： 10.3892/or.2012.2001

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REIC/Dkk-3-encoding adenoviral vector as a potentially effective therapeutic agent for bladder cancer

Takeshi Hirata, Masami Watanabe, Haruki Kaku, Yasuyuki Kobayashi, Hiroshi Yamada, Masakiyo Sakaguchi, Kohji Takei, Nam-Ho Huh, Yasutomo Nasu, Hiromi Kumon

INTERNATIONAL JOURNAL OF ONCOLOGY   41 ( 2 )   559 - 564   2012.8

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Bladder cancer is one of the most common urogenital malignancies. The intravesical instillation of anticancer agents is an attractive strategy to treat a superficial lesion or floating/disseminated cancer cells after transurethral operation. An adenovirus carrying REIC/Dkk-3, a tumor suppressor gene (Ad-REIC), exhibits cancer-specific apoptotic effects in various types of cancer cells. The aim of the present study was to examine the potential of Ad-REIC as a therapeutic agent for bladder cancer. KK47 and RT4 human bladder cancer cells were sensitive to the Ad-REIC treatment for apoptosis induction, but some human bladder cancer cell lines (T24, J82 and TccSup) were resistant. Significant cell growth inhibition was observed when these resistant cancer cell lines were treated with Ad-REIC in a condition of floating cells, which is clinically observed after transurethral operation and becomes a cause of intravesical cancer dissemination. The therapeutic potential of Ad-REIC for the treatment of multidrug-resistant bladder cancer was investigated. The adriamycin-resistant KK47 bladder cancer cells (KK47/ADM), which also present multidrug resistance, showed induction of significant apoptosis following Ad-REIC treatment. The Ad-REIC treatment induced downregulation of P-glycoprotein in KK47/ADM cells and restored the sensitivity to doxorubicin (adriamycin). Ad-REIC suppressed P-glycoprotein expression in a c-Jun-NH2-kinase (JNK)-dependent manner. Therefore, the current study indicated two therapeutic aspects of the Ad-REIC agent against human bladder cancer cells, as an apoptosis inducer/cell growth inhibitor and as a sensitizer of chemotherapeutic agents in multidrug-resistant cancer cells. The intravesical instillation of Ad-REIC could be an attractive therapeutic method in human bladder cancer where the treatment of superficial lesions and floating/disseminated or multidrug-resistant cancer cells is necessary.

DOI： 10.3892/ijo.2012.1503

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S100A8およびS100A9タンパク質の新規受容体の探索とそのがん進展における役割(Identification of novel receptors for pro-inflammatory S100A8/A9 proteins and their potential roles in tumor progression)

阪口 政清, 日比野 利彦, 村田 等, 井上 裕介, 山本 健一, 片岡 健, 許 南浩

日本癌学会総会記事   71回   59 - 59   2012.8

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Expression pattern of REIC/Dkk-3 in mouse squamous epithelia

K. Kataoka, G. Du, N. Maehara, H. Murata, M. Sakaguchi, N. Huh

CLINICAL AND EXPERIMENTAL DERMATOLOGY   37 ( 4 )   428 - 431   2012.6

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Language：English   Publisher：WILEY-BLACKWELL  

The REIC/Dkk (reduced expression in immortalized cells/Dickkopf-3) gene was originally identified as a tumour-suppressor gene with reduced expression in immortalized cells, cancer-cell lines and tumour tissues. Of the four members of the Dkk family, the REIC/Dkk-3 protein is unique in terms of DNA sequence, expression profiles and biological functions. In this study, we investigated and compared the expression patterns of the REIC/Dkk-3 protein in mouse squamous epithelia. Expression of REIC/Dkk-3 in the back skin was localized to the upper layer of the interfollicular epidermis, and the inner root sheath of hair follicles. Expression of REIC/Dkk-3 was detected in the ear skin, oral mucosa, tongue, oesophagus, uterine cervix, footpad and tail skin, but not in the cornea. Interestingly, expression was localized to the upper layers of these epithelial tissues. The physiological function of REIC/Dkk-3 is still unclear, but our detailed observation highlight its unique expression pattern in squamous epithelia.

DOI： 10.1111/j.1365-2230.2011.04301.x

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S100A7 promotes the migration and invasion of osteosarcoma cells via the receptor for advanced glycation end products

Ken Kataoka, Tomoyuki Ono, Hitoshi Murata, Mika Morishita, Ken-Ichi Yamamoto, Masakiyo Sakaguchi, Nam-Ho Huh

ONCOLOGY LETTERS   3 ( 5 )   1149 - 1153   2012.5

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Osteosarcoma is the most common malignant tumor of bone in childhood and adolescence. Despite intensive research for new therapies, the outcome in patients with metastasis remains extremely poor. S100 proteins are involved in the proliferation, cell cycle progression and metastasis of numerous malignant tumors, including osteosarcoma. In the present study, we identified S100A7 as a candidate to promote the migration of osteosarcoma cells. S100A7 promoted the migration and invasion of osteosarcoma cells as assayed in vitro. An in vitro pull-down assay revealed the binding of the recombinant S100A7 protein with its putative receptor, the receptor for advanced glycation end products (RAGE). The downregulation of RAGE by a specific siRNA markedly suppressed the migration and invasion of osteosarcoma cells. Furthermore, the matrix metalloproteinase activity of osteosarcoma cells was enhanced by S100A7 and suppressed by the downregulation of RAGE. These results indicate that S100A7 promotes the migration and invasion of osteosarcoma cells through RAGE. The S100A7-RAGE axis may thus be a new target for preventing the invasion and/or metastasis of osteosarcoma.

DOI： 10.3892/ol.2012.612

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PS-109-6 REIC/Dickkopf-3 shows anti-proliferative effect for non-small cell lung cancer cells with negative phosphorylated-Akt status

Tanaka Norimitsu, Toyooka Shinichi, Watanabe Masami, Soh Junichi, Sakaguchi Masakiyo, Tsukuda Kazunori, Asano Hiroaki, Shien Kazuhiko, Furukawa Masashi, Maki Yuho, Muraoka Takayuki, Huh Nam-ho, Nasu Yasutomo, Kumon Hiromi, Miyoshi Shinichiro

Journal of Japan Surgical Society   113 ( 2 )   710 - 710   2012.3

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Partial sensitization of human bladder cancer cells to a gene-therapeutic adenovirus carrying REIC/Dkk-3 by downregulation of BRPK/PINK1

Yu Jin, Hitoshi Murata, Masakiyo Sakaguchi, Ken Kataoka, Masami Watanabe, Yasutomo Nasu, Hiromi Kumon, Nam-Ho Huh

ONCOLOGY REPORTS   27 ( 3 )   695 - 699   2012.3

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REIC/Dkk-3 is a tumor suppressor gene that was first identified as a gene downregulated in association with immortalization of normal human fibroblasts. We have demonstrated that an adenovirus carrying REIC/Dkk-3 (Ad-REIC) showed a tumor-specific killing effect on a wide range of cancers. However, some human cancers, bladder cancers in particular, are resistant to Ad-REIC. In this study, we investigated the combination effect of downregulation of BRPK/PINK1 (PINK1) and Ad-REIC on bladder cancer cells. Five bladder cancer cell lines among six cell lines examined were resistant to Ad-REIC. Among the cell lines, the resistance of two cell lines was probably due to low infection efficiency of the adenovirus. PINK1-specific siRNA remarkably downregulated Bcl-x(L) and TRAP1 proteins and upregulated BAX protein expression. Finally, downregulation of PINK1 partially sensitized the other three cell lines that were resistant to Ad-REIC. This sensitization was associated with increasing production of reactive oxygen species (ROS). These results indicate that PINK1 is one of the key molecules for the mitochondrial protection system and that PINK1 can be a new target molecule to sensitize bladder cancer cells that are resistant to Ad-REIC.

DOI： 10.3892/or.2011.1543

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Preclinical Safety and Efficacy of in Situ REIC/Dkk-3 Gene Therapy for Prostate Cancer

Keiichiro Kawauchi, Masami Watanabe, Haruki Kaku, Peng Huang, Kasumi Sasaki, Masakiyo Sakaguchi, Kazuhiko Ochiai, Nam-ho Huh, Yasutomo Nasu, Hiromi Kumon

ACTA MEDICA OKAYAMA   66 ( 1 )   7 - 16   2012.2

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Language：English   Publisher：OKAYAMA UNIV MED SCHOOL  

The preclinical safety and therapeutic efficacy of adenoviral vectors that express the REIC/Dkk-3 tumor suppressor gene (Ad-REIC) was examined for use in prostate cancer gene therapy. The Ad-human (h) and mouse (m) REIC were previously demonstrated to induce strong anti-cancer effects in vitro and in vivo, and we herein report the results of two in vivo studies. First, intra-tumor Ad-hREIC administration was examined for toxicity and therapeutic effects in a subcutaneous tumor model using the PC3 prostate cancer cell line. Second, intra-prostatic Ad-mREIC administration was tested for toxicity in normal mice. The whole-body and spleen weights, hematological and serum chemistry parameters, and histological evaluation of tissues from throughout the body were analyzed. Both experiments indicated that there was no significant difference in the examined parameters between the Ad-REIC-treated group and the control (PBS- or Ad-LacZ-treated) group. In the in vitro analysis using PC3 cells, a significant apoptotic effect was observed after Ad-hREIC treatment. Confirming this observation, the robust anti-tumor efficacy of Ad-hREIC was demonstrated in the in vivo subcutaneous prostate cancer model. Based on the results of these preclinical experiments, we consider the adenovirus-mediated REIC/Dkk-3 in situ gene therapy to be safe and useful for the clinical treatment of prostate cancer.

DOI： 10.18926/AMO/48076

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C型ボツリヌス毒素変換ファージのゲノム情報を基盤とした偽溶原性メカニズムの解析

阪口義彦, OLIVA Maria A, MARTIN‐GALIANO Antonio J, ANDREU Jose M, 阪口政清, 内山淳平, 小椋義俊, 山本由弥子, 鈴木智典, 織田華絵, 津田千秋, 松崎茂展, 林哲也, 小熊惠二

日本分子生物学会年会プログラム・要旨集(Web)   35th   WEB ONLY 3W3III-2   2012

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TRAF6とSARM1によるPINK1のユビキチン化はマイトファジーの誘導に必須である

村田等, 阪口政清, 片岡健, HUH Nam‐ho

日本分子生物学会年会プログラム・要旨集(Web)   35th   4P-0506 (WEB ONLY)   2012

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多機能受容体RAGEの下流信号伝達機構の解明(TIRAP is a critical transducer of RAGE-mediated inflammatory signaling)

阪口 政清, 村田 等, 山本 健一, 阪口 義彦, 本山 晃, 日比野 利彦, 片岡 健, 許 南浩

日本癌学会総会記事   70回   225 - 225   2011.9

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Language：English   Publisher：日本癌学会  

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PINK1発現抑制によるミトコンドリア機能不全はREIC/Dkk-3のアポトーシス誘導効果を増強する(Mitochondrial malfunction by PINK1 knockdown augments apoptosis induced by adenovirus carrying REIC/Dkk-3)

村田 等, 金 玉, 阪口 政清, 片岡 健, 許 南浩

日本癌学会総会記事   70回   51 - 51   2011.9

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S100A7による骨肉腫細胞の遊走・浸潤の多機能受容体RAGEを介した制御機構(Promotion of migration and invasion of osteosarcoma cells by S100A7 through RAGE)

片岡 健, 阪口 政清, 小野 智之, 森下 美加, 山本 健一, 村田 等, 許 南浩

日本癌学会総会記事   70回   79 - 79   2011.9

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Tumor suppressor REIC/Dkk-3 interacts with the dynein light chain, Tctex-1

Kazuhiko Ochiai, Masami Watanabe, Hideo Ueki, Peng Huang, Yasuyuki Fujii, Yasutomo Nasu, Hirofumi Noguchi, Takeshi Hirata, Masakiyo Sakaguchi, Nam-ho Huh, Yuji Kashiwakura, Haruki Kaku, Hiromi Kumon

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   412 ( 2 )   391 - 395   2011.8

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

REIC/Dkk-3 is a member of the Dickkopf family proteins known as Wnt-antagonists, and REIC/Dkk-3 expression is downregulated in a broad range of cancer types. REIC/Dkk-3 acts as a tumor suppressor in multiple cancer cell lines by inducing apoptosis through endoplasmic reticulum (ER) stress signaling. However, the intracellular interaction partners of REIC/Dkk-3 have not been fully elucidated. By employing yeast two-hybrid screening, we identified the human dynein light chain, Tctex-1, as a novel interaction partner of REIC/Dkk-3. We further disclosed that the interaction involves the 136-157 amino acid region of REIC/Dkk-3 by using the mammalian two-hybrid system. Interestingly, this binding region of REIC/Dkk-3 with Tctex-1 contains an amino acid sequence motif [-(E) under bar -X-(G) under bar-(R) under bar-(R) under bar -X-(H) under bar-] which was previously reported as the Tctex-1 binding domain of dynein intermediate chain (DIC). Immunocytochemistry demonstrated that both REIC/Dkk-3 and Tctex-1 were localized around the ER of human fibroblasts, and the similar distribution pattern of the proteins suggests that their interaction occurs around the ER. This is the first study showing the interaction of a Dickkopf family protein with a dynein motor complex protein. The link between REIC/Dkk-3 and Tctex-1 may be of significance for understanding the molecular functions of the proteins in ER stress signaling and intracellular dynein motor dynamics, respectively. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.07.109

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Epigenetic Silencing of MicroRNA-34b/c Plays an Important Role in the Pathogenesis of Malignant Pleural Mesothelioma

Takafumi Kubo, Shinichi Toyooka, Kazunori Tsukuda, Masakiyo Sakaguchi, Takuya Fukazawa, Junichi Soh, Hiroaki Asano, Tsuyoshi Ueno, Takayuki Muraoka, Hiromasa Yamamoto, Yasutomo Nasu, Takumi Kishimoto, Harvey I. Pass, Hideki Matsui, Nam-ho Huh, Shinichiro Miyoshi

CLINICAL CANCER RESEARCH   17 ( 15 )   4965 - 4974   2011.8

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Purpose: Malignant pleural mesothelioma (MPM) is an aggressive tumor with a dismal prognosis. Unlike other malignancies, TP53 mutations are rare in MPM. Recent studies have showed that altered expression of microRNA (miRNA) is observed in human malignant tumors. In this study, we investigated the alterations of miR-34s, a direct transcriptional target of TP53, and the role of miR-34s on the pathogenesis of MPM.
Experimental Design: Aberrant methylation and expression of miR-34s were examined in MPM cell lines and tumors. miR-34b/c was transfected to MPM cells to estimate the protein expression, cell proliferation, invasion, and cell cycle.
Results: Aberrant methylation was present in 2 (33.3%) of 6 MPM cell lines and 13 (27.7%) of 47 tumors in miR-34a and in all 6 MPM cell lines (100%) and 40 (85.1%) of 47 tumors in miR-34b/c. Expression of miR-34a and 34b/c in all methylated cell lines was reduced and restored with 5-aza-2'-deoxycytidine treatment. Because epigenetic silencing was the major event in miR-34b/c, we investigated the functional role of miR-34b/c in MPM. miR-34b/c-transfected MPM cells with physiologic miR-34b/c expression exhibited antiproliferation with G(1) cell cycle arrest and suppression of migration, invasion, and motility. The forced overexpression of miR-34b/c, but not p53, showed a significant antitumor effect with the induction of apoptosis in MPM cells.
Conclusions: We show that the epigenetic silencing of miR-34b/c by methylation is a crucial alteration and plays an important role in the tumorigenesis of MPM, suggesting potential therapeutic options for MPM. Clin Cancer Res; 17(15); 4965-74. (C)2011 AACR.

DOI： 10.1158/1078-0432.CCR-10-3040

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TIRAP, an Adaptor Protein for TLR2/4, Transduces a Signal from RAGE Phosphorylated upon Ligand Binding

Masakiyo Sakaguchi, Hitoshi Murata, Ken-ichi Yamamoto, Tomoyuki Ono, Yoshihiko Sakaguchi, Akira Motoyama, Toshihiko Hibino, Ken Kataoka, Nam-ho Huh

PLOS ONE   6 ( 8 )   23132 - 23132   2011.8

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The receptor for advanced glycation end products (RAGE) is thought to be involved in the pathogenesis of a broad range of inflammatory, degenerative and hyperproliferative diseases. It binds to diverse ligands and activates multiple intracellular signaling pathways. Despite these pivotal functions, molecular events just downstream of ligand-activated RAGE have been surprisingly unknown. Here we show that the cytoplasmic domain of RAGE is phosphorylated at Ser391 by PKCf upon binding of ligands. TIRAP and MyD88, which are known to be adaptor proteins for Toll-like receptor-2 and -4 (TLR2/4), bound to the phosphorylated RAGE and transduced a signal to downstream molecules. Blocking of the function of TIRAP and MyD88 largely abrogated intracellular signaling from ligand-activated RAGE. Our findings indicate that functional interaction between RAGE and TLRs coordinately regulates inflammation, immune response and other cellular functions.

DOI： 10.1371/journal.pone.0023132

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Multiple functions of PINK1 at different intracellular locations Beyond neurodegenerative diseases

Hitoshi Murata, Masakiyo Sakaguchi, Ken Kataoka, Nam-ho Huh

CELL CYCLE   10 ( 10 )   1518 - 1519   2011.5

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Language：English   Publisher：LANDES BIOSCIENCE  

DOI： 10.4161/cc.10.10.15445

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Markedly elevated serum levels of calcium-binding S100A8/A9 proteins in psoriatic arthritis are due to activated. monocytes/macrophages

Seiko Aochi, Kazuhide Tsuji, Masakiyo Sakaguchi, Namho Huh, Tatsuya Tsuda, Kiyofumi Yamanishi, Mayumi Komine, Keiji Iwatsuki

JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY   64 ( 5 )   879 - 887   2011.5

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Background: Serum levels of S100A8/A9 may correlate with disease activity in psoriasis.
Objective: We sought to elucidate the association of serum levels of S100A8/A9 heterodimers with the clinical subtypes of psoriasis and the major cell source.
Methods: Serum samples were collected from patients with psoriasis vulgaris (n = 30), psoriatic arthritis (PA) (n = 16),. pustular psoriasis (n = 24), and atopic dermatitis (n = 14) and from healthy control subjects (n = 21). Serum concentrations of S100A8/A9 were measured, and the expression levels were examined in psoriatic lesions. The messenger RNA levels were quantified in circulating monocytes and neutrophils.
Results: Serum levels of S100A8/A9 were significantly increased in all subtypes of psoriasis as compared with healthy controls and atopic dermatitis. Among the psoriatic subtypes, PA and pustular psoriasis showed remarkably high concentrations of S100A8/A9 heterodimers. The higher serum levels were associated with the presence of articular symptoms, but not significantly correlated with body surface areas of psoriatic lesions. S100A8 was expressed by both keratinocytes and infiltrating mononuclear cells, whereas S100A9 was predominantly expressed by neutrophils. The expression levels of S100A8 and S100A9 messenger RNA in monocytes were increased by approximately 2.25- and 1.91-fold in PA, respectively, whereas no significant increase was observed in psoriasis vulgaris and pustular psoriasis.
Limitations: Difficulty in acquisition of clinical and laboratory samples in untreated patients, and of a sufficient number of subjects, were limitations.
Conclusions: Although serum levels of S100A8/A9 were increased in all types of psoriasis examined, patients with PA had higher levels of S100A8/A9, probably because of an activated monocyte/macrophage system. (J Am Acad Dermatol 2011;64:879-87.)

DOI： 10.1016/j.jaad.2010.02.049

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Intraperitoneal administration of an adenovirus vector carrying REIC/Dkk-3 suppresses peritoneal dissemination of scirrhous gastric carcinoma

Swe Swe Than, Ken Kataoka, Masakiyo Sakaguchi, Hitoshi Murata, Fernando Abarzua, Chika Taketa, Gang Du, Masakazu Yashiro, Kazuyoshi Yanagihara, Yasutomo Nasu, Hiromi Kumon, Nam-Ho Huh

ONCOLOGY REPORTS   25 ( 4 )   989 - 995   2011.4

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Expression levels of the novel tumor suppressor gene REIC/Dkk-3 are reduced in many human cancers. We have previously showed that an adenovirus vector carrying REIC/Dkk-3 (Ad-REIC) induced apoptosis of cancer cells selectively and exerted bystander antitumor effects via ER stress. We examined possible effects of Ad-REIC in a peritoneal dissemination model of scirrhous gastric carcinoma (SGC). Among various types of gastric cancer, SGC continues to be associated with the worst prognosis due to a high incidence of metastases in the peritoneal cavity. We found that a single intraperitoneal injection of Ad-REIC suppressed tumor dissemination and disease progression. Immunomodulation by Ad-REIC led to recruitment of natural killer cells inside tumor nodules. We conclude that Ad-REIC gene therapy may be a potential tool in combinatorial approaches to achieve curative effects in SGC.

DOI： 10.3892/or.2011.1149

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Identification of EMMPRIN as a novel receptor for S100A9: Its involvement in melanoma metastasis

T. Hibino, S. Miyamoto, M. Sakaguchi, A. Motoyama, M. Yamamoto, R. Tsuboi, N. Huh

JOURNAL OF INVESTIGATIVE DERMATOLOGY   131   S126 - S126   2011.4

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：NATURE PUBLISHING GROUP  

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Expression of REIC/Dkk-3 in normal and hyperproliferative epidermis

Gang Du, Ken Kataoka, Masakiyo Sakaguchi, Fernando Abarzua, Swe Swe Than, Hiroyuki Sonegawa, Teruhiko Makino, Tadamichi Shimizu, Nam-Ho Huh

EXPERIMENTAL DERMATOLOGY   20 ( 3 )   273 - 277   2011.3

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Dickkopf (Dkk) family members are known as Wnt modulators involved in the development, cell growth/differentiation and cancer. REIC/Dkk-3, which does not interfere with Wnt signalling, has been proposed to be a tumor suppressor gene, but its physiological function has remained unclear. In this study, we analysed the expression of REIC/Dkk-3 in normal interfollicular epidermis (IFE) and hyperproliferative epidermis. REIC/Dkk-3 was expressed in human and mouse IFE, being localized at the interface of upper spinous layer and granular layer. Skin cancer cell lines lost REIC/Dkk-3 expression as reported previously. When we analysed patient samples, REIC/Dkk-3 expression was down-regulated in the hyperproliferative epidermis including skin cancers and non-cancerous proliferative diseases. REIC/Dkk-3 expression was also suppressed in the regenerative and inflammative epidermis of model mice. These findings will certainly contribute to the extension of studies on REIC/Dkk-3.

DOI： 10.1111/j.1600-0625.2010.01244.x

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REIC/Dkk-3の扁平上皮における発現とその制御因子の探索

片岡 健, 阪口 政清, 杜 剛, 前原 奈都美, 村田 等, 許 南浩

組織培養研究   30 ( 1 )   99 - 99   2011.3

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A New Cytosolic Pathway from a Parkinson Disease-associated Kinase, BRPK/PINK1 ACTIVATION OF AKT VIA MTORC2

Hitoshi Murata, Masakiyo Sakaguchi, Yu Jin, Yoshihiko Sakaguchi, Jun-ichiro Futami, Hidenori Yamada, Ken Kataoka, Nam-ho Huh

JOURNAL OF BIOLOGICAL CHEMISTRY   286 ( 9 )   7182 - 7189   2011.3

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Accumulating evidence indicates that dysfunction of mitochondria is a common feature of Parkinson disease. Functional loss of a familial Parkinson disease-linked gene, BRPK/PINK1 (PINK1), results in deterioration of mitochondrial functions and eventual neuronal cell death. A mitochondrial chaperone protein has been shown to be a substrate of PINK1 kinase activity. In this study, we demonstrated that PINK1 has another action point in the cytoplasm. Phosphorylation of Akt at Ser-473 was enhanced by overexpression of PINK1, and the Akt activation was crucial for protection of SH-SY5Y cells from various cytotoxic agents, including oxidative stress. Enhanced Akt phosphorylation was not due to activation of phosphatidylinositol 3-kinase but due to activation of mammalian target of rapamycin complex 2 (mTORC2) by PINK1. Rictor, a specific component of mTORC2, was phosphorylated by overexpression of PINK1. Furthermore, overexpression of PINK1 enhanced cell motility. These results indicate that PINK1 exerts its cytoprotective function not only in mitochondria but also in the cytoplasm through activation of mTORC2.

DOI： 10.1074/jbc.M110.179390

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正常皮膚におけるREIC/Dkk‐3の発現制御因子の検索

前原奈都美, 片岡健, DU Gang, 村田等, 山本健一, 阪口政清, HUH Nam‐Ho

日本分子生物学会年会プログラム・要旨集(Web)   34th   1P-0254 (WEB ONLY)   2011

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S100タンパク質受容体の結合解析

森下美加, 村田等, 山本健一, 片岡健, 阪口政清, HUH Nam‐Ho

日本分子生物学会年会プログラム・要旨集(Web)   34th   4P-0293 (WEB ONLY)   2011

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A novel tumor suppressor, REIC/Dkk-3 gene identified by our in vitro transformation model of normal human fibroblasts works as a potent therapeutic anti-tumor agent

Masakiyo Sakaguchi, Nam-Ho Huh, Masayoshi Namba

Advances in Experimental Medicine and Biology   720   209 - 215   2011

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Reduced Expression in Immortalized Cell (REIC) was cloned by subtractive hybridization method as a gene whose expression is reduced in many human immortalized and neoplastic tumor cells. The REIC, when over-expressed by an adenovirus (Ad-REIC), exhibited a dramatic therapeutic effect on a wide variety of human cancers through a mechanism triggered by ER-stress-mediated JNK activation. In addition to this direct effect on cancer cells, Ad-REIC exerted another cytotoxicity on human cancers, an indirect host-mediated effect due to overproduction of IL-7 by mis-targeted normal cells. This "one-bullet two-arms" finding may lead to a powerful new therapeutic approach to the treatment of human cancers. © 2011 Springer Science+Business Media, LLC.

DOI： 10.1007/978-1-4614-0254-1_17

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Expression pattern of REIC/Dkk-3 in various cell types and the implications of the soluble form in prostatic acinar development

Kai Zhang, Masami Watanabe, Yuji Kashiwakura, Shun-Al Li, Kohei Edamura, Peng Huang, Ken Yamaguchi, Yasutomo Nasu, Yasuyuki Kobayashi, Masakiyo Sakaguchi, Kazuhiko Ochiai, Hiroshi Yamada, Kohji Takei, Hideo Ueki, Nam-Ho Huh, Ming Li, Haruki Kaku, Yanqun Na, Hiromi Kumon

INTERNATIONAL JOURNAL OF ONCOLOGY   37 ( 6 )   1495 - 1501   2010.12

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Language：English   Publisher：SPANDIDOS PUBL LTD  

The tumor suppressor REIC/Dkk-3 is a secretory protein which was originally identified to be downregulated in human immortalized cells In the present study, we investigated the expression pattern of REIC/Dkk-3 in various cell types to characterize its physiological functions We first examined the expression level of REIC/Dkk-3 in a broad range of cancer cell types and confirmed that it was significantly downregulated in all of the cell types We also examined the tissue distribution pattern in a variety of normal mouse organs Ubiquitous REIC/Dkk-3 protein expression was observed in the organs The expression was abundant in the liver, heart and brain tissue, but was absent in the spleen and peripheral blood mononuclear cells The immunohistochemical analyses revealed that the subcellular localization of REIC/Dkk-3 had a punctate pattern around the nucleus, indicating its association with secretory vesicles In cancer cells stably transfected with REIC/Dkk-3 the protein was predominantly localized to the endoplasmic reticulum (ER) under observation with confocal microscopy Because REIC/ Dkk-3 was found to be abundantly expressed in the acinar epithelial cells of the mouse prostate, we analyzed the effects of recombinant REIC/Dkk-3 protein on the acinar morphogenesis of RWPE-1 cells, which are derived from human normal prostate epithelium Statistically significant acinar growth was observed in the culture condition with 10 mu g/m1 REIC/Dkk-3 protein, implicating the soluble form m prostatic acinar development Current results suggest that REIC/Dkk-3 may play a role in regulating the morphological process of normal tissue architecture through an autocrine and/or paracrine manner

DOI： 10.3892/ijo_00000802

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KLF11とKLF15によるUCP1の発現制御

山本 健一, 阪口 政清, 片岡 健, 許 南浩

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集   83回・33回   2P - 0822   2010.12

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BRPK/PINK1のmTORC2経路活性化を介したがん進展への寄与(BRPK/PINK1 promotes tumor progression through activation of mTORC2 pathway)

村田 等, 阪口 政清, 金 玉, 片岡 健, 許 南浩

日本生化学会大会・日本分子生物学会年会合同大会講演要旨集   83回・33回   4P - 0934   2010.12

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Internalization of REIC/Dkk-3 protein by induced pluripotent stem cell-derived embryoid bodies and extra-embryonic tissues

Ken Kataoka, Masakiyo Sakaguchi, Kun Peng Li, Chika Taketa, Ken-Ichi Yamamoto, Gang Du, Hiroaki Funahashi, Hitoshi Murata, Nam-Ho Huh

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE   26 ( 6 )   853 - 859   2010.12

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REIC/Dkk-3 was first identified as a down-regulated gene in a number of human immortalized cells and human tumor-derived cell lines. Overexpression of the REIC/Dkk-3 gene using an adenovirus vector (Ad-REIC) has showed a potent selective therapeutic effect on various human cancers through induction of ER stress. Furthermore, we recently showed that Ad-REIC has an indirect host-mediated anti-tumor activity by induction of IL-7. However, the physiological function of REIC/Dkk-3 is still unclear. As a first step to study the possible receptor(s) for secreted REIC/Dkk-3, we analyzed the internalization of Cy3-labeled recombinant REIC/Dkk-3 protein. Among the cell lines screened, mouse induced pluripotent stem (iPS) cells showed a unique pattern of internalization. The internalization was observed in peripheral cells of spherical colonies formed spontaneously, but not in undifferentiated iPS cells. When we analyzed embryoid bodies (EBs) derived from iPS cells, REIC/Dkk-3 protein was internalized specifically by differentiated cells located at the periphery of EBs. Interestingly, Dkk-1 was internalized by undifferentiated cells at the center of the EBs. When developmental tissue was analyzed, internalization of REIC/Dkk-3 protein was strictly limited to extra-embryonic tissue, such as the trophectoderm layer of 4.5 days post-coitus (dpc) blastocysts and the chorionic membrane at 16.5 dpc. The mechanism of the internalization was confirmed to be endocytosis. These findings will contribute to knowledge on the interaction of REIC/Dkk-3 with a possible receptor(s).

DOI： 10.3892/ijmm_00000534

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Transcriptional regulation of a brown adipocyte-specific gene, UCP1, by KLF11 and KLF15

Ken-ichi Yamamoto, Masakiyo Sakaguchi, Reinhold J. Medina, Aya Niida, Yoshihiko Sakaguchi, Masahiro Miyazaki, Ken Kataoka, Nam-ho Huh

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   400 ( 1 )   175 - 180   2010.9

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Several growth factors and transcription factors have been reported to play important roles in brown adipocyte differentiation and modulation of thermogenic gene expression, especially the expression of UCP1. In this study, we focused on KLF11 and KLF15, which were expressed highly in brown adipose tissue. Our data demonstrated that KLF11 and KLF15 interacted directly with the UCP1 promoter using GC-box and GT-boxes, respectively. Co-transfection of KLF11 and KLF15 in the mesenchymal stem cell line muBM3.1 during brown adipocyte differentiation enhanced the expression level of UCP1. KLF11, but not KLF15, was essential for UCP1 expression during brown adipocyte differentiation of muBM3.1. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.08.039

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アデノウイルス受容体(CAR;CXADR)の固形癌細胞の増殖制御における役割の解析(Biological role of Coxsackievirus and Adenovirus Receptor (CXADR) in cancer cell proliferation)

小屋 恵理子, 阪口 政清, 松井 誠, 冨田 勇樹, 許 南浩, 近藤 英作

日本癌学会総会記事   69回   268 - 268   2010.8

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スキルス胃がん腹膜播種へのREIC/Dkk-3遺伝子治療の効果(REIC/Dkk-3 gene therapy suppresses peritoneal dissemination of scirrhous gastric carcinoma)

片岡 健, タン・スエスエ, 阪口 政清, アバルスァ・フェルナンド, 村田 等, 八代 正和, 那須 保友, 公文 裕巳, 許 南浩

日本癌学会総会記事   69回   252 - 253   2010.8

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新規がん抑制遺伝子REIC/Dkk-3の増殖性皮膚疾患での発現減少(Reduced expression of REIC/Dkk-3, a putative tumor suppressor gene, in hyperproliferative epidermis)

杜 剛, 片岡 健, 阪口 政清, 牧野 輝彦, 清水 忠道, 許 南浩

日本癌学会総会記事   69回   316 - 316   2010.8

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マウス骨髄間葉系幹細胞の褐色脂肪分化をKLF11およびKLF15は促進する

片岡健, 山本健一, 阪口政清, HUH Nam‐ho

硬組織再生生物学会学術大会・総会プログラム・抄録集   19th   40   2010.7

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Down-regulation of BiP/GRP78 sensitizes resistant prostate cancer cells to gene-therapeutic overexpression of REIC/Dkk-3

Ryuta Tanimoto, Masakiyo Sakaguchi, Fernando Abarzua, Ken Kataoka, Kaoru Kurose, Hitoshi Murata, Yasutomo Nasu, Hiromi Kumon, Nam-Ho Huh

INTERNATIONAL JOURNAL OF CANCER   126 ( 7 )   1562 - 1569   2010.4

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We have recently shown that an adenovirus carrying REIC/Dkk-3 (Ad-REIC) exhibits a potent tumor-specific cell-killing function for various human cancers. It has also become evident that some human cancers are resistant to Ad-REIC-induced apoptosis. The aim of the present study was to determine the molecular mechanisms of resistance to Ad-REIC. First, we isolated resistant clones from a human prostate cancer cell line, PC3, after repeated exposure to Ad-REIC. Infection efficiency of the adenovirus vector and expression level of REIC/Dkk-3 in the resistant clones were similar to those in the parental PC3 cells. By screening for alteration in levels and functional status of proteins involved in Ad-REIC-induced apoptosis, we found that BiP/GRP78, an ER-residing chaperone protein, was expressed at higher levels consistently among resistant cells. Expression levels of BiP and rates of apoptosis induced by Ad-REIC were inversely correlated. Down-regulation of BiP with siRNA sensitized the resistant cells to Ad-REIC in vivo as well as in culture. These results indicate that BiP is a major determinant of resistance to Ad-REIC-induced apoptosis. Thus BiP is useful for diagnosis of inherent and acquired resistance of cancers and also as a target molecule to overcome resistance to the gene therapeutic Ad-REIC.

DOI： 10.1002/ijc.24764

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分化誘導したマウスES細胞及びiPS細胞におけるCy3標識REIC/Dkk‐3タンパク質のエンドサートーシスによる取り込み

片岡健, 阪口政清, LI Kun Peng, DU Gang, 武田千佳, 舟橋弘晃, 村田等, HUH Nam‐Ho

組織培養研究   29 ( 1 )   74 - 74   2010.3

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褐色脂肪細胞分化におけるKLF11とKLF15の役割

山本健一, 阪口政清, MEDINA Reinhold, 新居田彩, 宮崎正博, 片岡健, HUH Nam‐Ho

組織培養研究   29 ( 1 )   82   2010.3

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関節症性乾癬におけるバイオマーカーとしての血清S100A8/A9蛋白

青地聖子, 辻和英, 阪口政清, 許南浩, 津田達也, 山西清文, 小宮根真弓, 岩月啓氏

日本皮膚科学会雑誌   120 ( 3 )   691   2010.3

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S100A11, a dual growth regulator of epidermal keratinocytes.

Sakaguchi M, Huh NH

Amino Acids   1111 - 1111   2010

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BRPK/PINK1のmTORC2経路活性化を介したがん浸潤への寄与(BRPK/PINK1 enhances invasiveness of cancer cells trough activation of mTORC2 pathway)

村田 等, 阪口 政清, 片岡 健, 許 南浩

日本癌学会総会記事   68回   205 - 205   2009.8

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Overexpression of REIC/Dkk-3 in Normal Fibroblasts Suppresses Tumor Growth via Induction of Interleukin-7

Masakiyo Sakaguchi, Ken Kataoka, Fernando Abarzua, Ryuta Tanimoto, Masami Watanabe, Hitoshi Murata, Swe Swe Than, Kaoru Kurose, Yuji Kashiwakura, Kazuhiko Ochiai, Yasutomo Nasu, Hiromi Kumon, Nam-ho Huh

JOURNAL OF BIOLOGICAL CHEMISTRY   284 ( 21 )   14236 - 14244   2009.5

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We previously showed that the tumor suppressor gene REIC/Dkk-3, when overexpressed by an adenovirus (Ad-REIC), exhibited a dramatic therapeutic effect on human cancers through a mechanism triggered by endoplasmic reticulum stress. Adenovirus vectors show no target cell specificity and thus may elicit unfavorable side effects through infection of normal cells even upon intra-tumoral injection. In this study, we examined possible effects of Ad-REIC on normal cells. We found that infection of normal human fibroblasts (NHF) did not cause apoptosis but induced production of interleukin (IL)-7. The induction was triggered by endoplasmic reticulum stress and mediated through IRE1 alpha, ASK1, p38, and IRF-1. When Ad-REIC-infected NHF were transplanted in a mixture with untreated human prostate cancer cells, the growth of the cancer cells was significantly suppressed. Injection of an IL-7 antibody partially abrogated the suppressive effect of Ad-REIC-infected NHF. These results indicate that Ad-REIC has another arm against human cancer, an indirect host-mediated effect because of overproduction of IL-7 by mis-targeted NHF, in addition to its direct effect on cancer cells.

DOI： 10.1074/jbc.M808002200

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BRPK/PINK1のmTORC2活性化を介したがん進展への寄与

村田 等, 阪口 政清, 片岡 健, 許 南浩

組織培養研究   28 ( 1 )   66 - 66   2009.3

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Language：Japanese   Publisher：日本組織培養学会  

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Immunological aspects of REIC/Dkk-3 in monocyte differentiation and tumor regression

Masami Watanabe, Yuji Kashiwakura, Peng Huang, Kazuhiko Ochiai, Junichiro Futami, Shun-Ai Li, Munenori Takaoka, Yasutomo Nasu, Masakiyo Sakaguchi, Nam-Ho Huh, Hiromi Kumon

INTERNATIONAL JOURNAL OF ONCOLOGY   34 ( 3 )   657 - 663   2009.3

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Language：English   Publisher：SPANDIDOS PUBL LTD  

The REIC/Dkk-3 gene has been reported to be a tumor suppressor and the expression is significantly down-regulated in a broad range of cancer cell types. The protein is secretory, but the physiological function remains unclear. This study demonstrated that recombinant REIC/Dkk-3 protein induced the differentiation of human CD14(+) monocytes into a novel cell type ((REIC/Dkx-3)Mo). (REIC/Dkk-3)Mo resembles immature dendritic cells generated with IL-4 and GM-CSF. Both these cell populations exhibit similar proportions of CD11c(+), CD40(+), CD86(+) and HLA-DR(+) cells and endocytic capacity, but (REIC/Dkk-3)Mo is negative for CD1a antigen. An analysis of the signal transducers and activators of transcription (STAT) pathways revealed that REIC/Dkk-3 induces phosphorylation of STAT 1 and STAT 3. Furthermore, intratumoral administration of REIC/Dkk-3 protein significantly suppressed tumor growth with CD11c(+) and CD8(+) (dendritic and killer T cell marker, respectively) cell accumulation and enhanced anticancer cytolytic activity of splenocytes. These data indicated a cytokine-like role of REIC/Dkk-3 protein in monocyte differentiation that might be exploited therapeutically.

DOI： 10.3892/ijo_00000191

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分化したマウスES細胞およびiPS細胞のCy3標識REIC/Dkk‐3タンパク質の取り込み

片岡健, 阪口政清, 李坤鵬, 武田千佳, 山本健一, 許南浩

再生医療   8   223   2009.2

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Language：Japanese  

J-GLOBAL

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REIC/Dkk-3 overexpression downregulates P-glycoprotein in multidrug-resistant MCF7/ADR cells and induces apoptosis in breast cancer

K. Kawasaki, M. Watanabe, M. Sakaguchi, Y. Ogasawara, K. Ochiai, Y. Nasu, H. Doihara, Y. Kashiwakura, N-h Huh, H. Kumon, H. Date

CANCER GENE THERAPY   16 ( 1 )   65 - 72   2009.1

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Language：English   Publisher：NATURE PUBLISHING GROUP  

The overexpression of reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), a tumor suppressor gene, induced apoptosis in human prostatic and testicular cancer cells. The aim of this study is to examine the potential of REIC/Dkk-3 as a therapeutic target against breast cancer. First, the in vitro apoptotic effect of Ad-REIC treatment was investigated in breast cancer cell lines and the adenovirus-mediated overexpression of REIC/Dkk-3 was thus found to lead to apoptotic cell death in a c-Jun-NH(2)-kinase (JNK) phosphorylaion-dependent manner. Moreover, an in vivo apoptotic effect and MCF/Wt tumor growth inhibition were observed in the mouse model after intratumoral Ad-REIC injection. As multidrug resistance (MDR) is a major problem in the chemotherapy of progressive breast cancer, the in vitro effects of Ad-REIC treatment were investigated in terms of the sensitivity of multidrug-resistant MCF7/ADR cells to doxorubicin and of the P-glycoprotein expression. Ad-REIC treatment in MCF7/ADR cells also downregulated P-glycoprotein expresssion through JNK activation, and sensitized its drug resistance against doxorubicin. Therefore, not only apoptosis induction but also the reversal of anticancer drug resistance was achieved using Ad-REIC. We suggest that REIC/Dkk-3 is a novel target for breast cancer treatment and that Ad-REIC might be an attractive agent against drug-resistant cancer in combination with conventional antineoplastic agents.

DOI： 10.1038/cgt.2008.58

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Suppression of Carbon Tetrachloride-Induced Liver Fibrosis by Transplantation of a Clonal Mesenchymal Stem Cell Line Derived From Rat Bone Marrow

Marhaen Hardjo, Masahiro Miyazaki, Masakiyo Sakaguchi, Takuro Masaka, Sukaeni Ibrahim, Ken Kataoka, Narn-ho Huh

CELL TRANSPLANTATION   18 ( 1 )   89 - 99   2009

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Language：English   Publisher：COGNIZANT COMMUNICATION CORP  

Transplantation of hepatocytes or bone marrow-derived cells has been shown to ameliorate liver fibrosis in animal models, but no direct comparison of relative efficiency has been made. The aim of this study was to compare the efficiency of a bone marrow-derived clonal mesenchymal stem cell line established by LIS (rBM25/S3) with that of its adipogenic or hepatogenic differentiation derivative for Suppression of rat liver fibrosis. After induction of differentiation of rBM25/S3 cells into adipogenic or hepatogenic cells in Culture, we intrasplenically transplanted the three types of cells into rats (3 x 10(7) cells/rat) before and 4 weeks after initiation of carbon tetrachloride treatment (1 ml/kg body weight twice a week for 8 weeks) to induce liver fibrosis. Undifferentiated rBM25/S3 cells were the most effective for suppression of liver fibrosis, followed by the adipogenic cells and hepatogenic cells. Expression levels of MMP-2 and MMP-9 were also highest in undifferentiated rBM25/S3 cells. These results indicate that bone marrow-derived clonal mesenchymal stem cell lines are useful for further mechanistic studies on cell-mediated suppression of liver fibrosis and that such cell lines will provide information on an appropriate cell source for transplantation therapy for cirrhosis.

DOI： 10.3727/096368909788237140

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REIC/Dkk-3 in normal fibroblasts suppresses tumor growth via induction of interleukin-7.

Chen J, Watanabe M, Huang P, Sakaguchi M, Ochiai K, Nasu Y, Ouchida M, Huh NH, Shimizu K, Kashiwakura Y, Kaku H, Kumon H

Int J Mol Med   24 ( 6 )   789 - 794   2009

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Mechanistic Analysis of Resistance to REIC/Dkk-3-induced Apoptosis in Human Bladder Cancer Cells

Tomoko Kobayashi, Masakiyo Sakaguchi, Ryuta Tanimoto, Fernando Abarzua, Mikiro Takaishi, Haruki Kaku, Ken Kataoka, Takashi Saika, Yasutomo Nasu, Masahiro Miyazaki, Hiromi Kumon, Nam-ho Huh

ACTA MEDICA OKAYAMA   62 ( 6 )   393 - 401   2008.12

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Language：English   Publisher：OKAYAMA UNIV MED SCHOOL  

We have recently shown that a new therapeutic modality using the REIC/Dkk-3 gene (Ad-REIC) is effective against various human cancers, including those of prostate, testis and breast origins. The aim of the present study was to examine the sensitivity of bladder cancers to Ad-REIC and to clarify the molecular mechanisms that determine sensitivity/resistance. We found that 2 human bladder cancer cell lines, T24 and J82, are resistant to Ad-REIC. In T24 and J82 cells, the ER stress response and activation of JNK were observed in a manner similar to that in the sensitive PC3 cells. Translocation of Bax to mitochondria occurred in PC3 cells but not in T24 and J82 cells. Bcl-2 was remarkably overexpressed in T24 and J82 compared with the expression levels in sensitive cell lines. Treatment of T24 and J82 cells with a Bcl-2 inhibitor sensitized the cells to Ad-REIC-induced apoptosis. The results indicate that some human bladder cancers are resistant to apoptosis induced by overexpression of REIC/Dkk-3, which is at least in part due to up-regrulation of Bcl-2. These results provide a basis for possible use of Bcl-2 as a marker of sensitive cancers and to try to sensitize resistant cancers to Ad-REIC by down-regrulation of Bcl-2.

DOI： 10.18926/AMO/30945

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The structure of S100A11 fragment explains a local structural change induced by phosphorylation

Takahide Kouno, Mineyuki Mizuguchi, Masakiyo Sakaguchi, Eiichi Makino, Yoshihiro Mori, Hiroyuki Shinoda, Tomoyasu Aizawa, Makoto Demur, Nam-Ho Huh, Keiichi Kawano

JOURNAL OF PEPTIDE SCIENCE   14 ( 10 )   1129 - 1138   2008.10

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Language：English   Publisher：JOHN WILEY & SONS LTD  

S100A11 protein is a member of the S100 family containing two EF-hand motifs. It undergoes phophorylation on residue T10 after cell stimulation such as an increase in Ca2+ concentration. Phosphorylated S100A11 I can be recognized by its target protein, nucleolin. Although S100A11 is initially expressed in the cytoplasm, it is transported to the nucleus by the action of nucleolin. In the nucleus, S100A11 suppresses the growth of keratinocytes through p21(CIP1/WAF1) activation and induces cell differentiation. Interestingly, the N-terminal fragment of S100A11 has the same activity as the full-length protein: i.e. it is phosphorylated in vivo and binds to nucleolin. In addition, this fragment leads to the arrest of cultured keratinocyte growth. We examined the solution structure of this fragment peptide and explored its structural properties before and after phosphorylation. In a trifluoroethanol solution, the peptide adopts the alpha-helical structure just as the corresponding region of the full-length S100A11. Phosphorylation induces a disruption of the N-capping conformation of the alpha-helix, and has a tendency to perturb its surrounding structure. Therefore, the phosphorylated threonine lies in the N-terminal edge of the alpha-helix. This local structural change can reasonably explain why the phosphorylation of a residue that is initially buried in the interior of protein allows it to be recognized by the binding partner. Copyright (C) 2008 European Peptide Society and John Wiley & Sons, Ltd.

DOI： 10.1002/psc.1050

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Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates

Hitoshi Murata, Junichiro Futami, Midori Kitazoe, Takayuki Yonehara, Hidetaka Nakanishi, Megumi Kosaka, Hiroko Tada, Masakiyo Sakaguchi, Yasuyuki Yagi, Masaharu Seno, Nam-ho Huh, Hidenori Yamada

JOURNAL OF BIOCHEMISTRY   144 ( 4 )   447 - 455   2008.10

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Language：English   Publisher：OXFORD UNIV PRESS  

The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)-glutathione conjugate. On screening of protein transduction, optimized PEI-glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.

DOI： 10.1093/jb/mvn087

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Derivation of hepato-pancreatic intermediate progenitor cells from a clonal mesenchymal stem cell line of rat bone marrow origin

Takuro Masaka, Masahiro Miyazaki, Gang Du, Marhaen Hardjo, Masakiyo Sakaguchi, Mikiro Takaishi, Ken Kataoka, Kazuhide Yamamoto, Nam-Ho Huh

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE   22 ( 4 )   447 - 452   2008.10

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Language：English   Publisher：PROFESSOR D A SPANDIDOS  

We have recently established a clonal mesenchymal stem cell line (rBM25/S3) from adult rat bone marrow. The cells have practically unlimited proliferation capacity (over 300 PDL), maintaining multipotency for differentiation. In the present study, we examined the potential for rBM25/S3 cells to differentiate into insulin-secreting cells. When cultured in the presence of HGF and FGF-4 on Matrigel, rBM25/S3 cells expressed genes specific to pancreatic B-cells as well as those specific to hepatocytes. They still maintained proliferation capacity with a doubling time of similar to 30 h. These hepato-pancreatic intermediate progenitor cells, but not the original undifferentiated rBM25/S3 cells, were induced by the overexpression of PDX-1 to produce significant amounts of insulin in a manner responding to glucose concentration in medium. The present culture system indicates a direction for further studies aimed at the realization of cell transplantation therapy for type I diabetes mellitus.

DOI： 10.3892/ijmm_00000041

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An N-terminal 78 amino acid truncation of REIC/Dkk-3 effectively induces apoptosis

Fernando Abarzua, Yuji Kashiwakura, Munenori Takaoka, Masami Watanabe, Kazuhiko Ochiai, Masakiyo Sakaguchi, Takao Iwawaki, Ryuta Tanimoto, Yasutomo Nasu, Nam-ho Huh, Hiromi Kumon

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   375 ( 4 )   614 - 618   2008.10

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Overexpression of REIC/Dkk-3 (a tumor suppressor gene) induces cancer cell apoptosis through endoplasmic reticulum (ER) stress. Therefore, the identification of the portion of REIC/Dkk-3 that causes ER stress may be essential for the development of cancer treatment based on REIC/Dkk-3. Here, we made several truncated forms of REIC/Dkk-3 and investigated their therapeutic potentials against prostate cancer. Among three truncated forms, a variant comprising the N-terminal 78 amino acid region of REIC/Dkk-3 ((1-78) REIC/Dkk-3) most strongly induced ER stress and apoptosis in human prostate cancer cells (PC3). For in vivo gene expression, we coupled a biodegradable polymer with naked DNA, which attained robust trans-gene expression in PC3-derived subcutaneous tumor. In therapeutic experiments, we demonstrated that multiple direct injections of polymer-conjugated (1-78)REIC/Dkk-3 plasmid provoke ER stress and significantly reduced the subcutaneous tumor volume compared with the control group. We suggest this non-viral strategy may be an effective alternative to viral gene therapy. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.08.079

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Down-regulation of Inhibition of Differentiation-1 via Activation of Activating Transcription Factor 3 and Smad Regulates REIC/Dickkopf-3-Induced Apoptosis

Yuji Kashiwakura, Kazuhiko Ochiai, Masami Watanabe, Fernando Abarzua, Masakiyo Sakaguchi, Munenori Takaoka, Ryuta Tanimoto, Yasutomo Nasu, Nam-ho Hub, Hiromi Kumon

CANCER RESEARCH   68 ( 20 )   8333 - 8341   2008.10

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Language：English   Publisher：AMER ASSOC CANCER RESEARCH  

REIC/Dickkopf-3 (Dkk-3), a tumor suppressor gene, has been investigated in gene therapy studies. Our previous study suggested that REIC/Dkk-3-induced apoptosis mainly resulted from phosphorylation of c-Jun-NH2 kinase (JNK) in prostate cancer cells. However, the precise mechanisms, especially the molecular mechanisms regulating JNK phosphorylation, remain unclear. In this study, we investigated the mechanisms participating in JNK phosphorylation in the context of a refractory cancer disease, malignant mesothelioma (MM). Adenovirus-mediated overexpression of REIC/Dkk-3 induced apoptosis mainly through JNK activation in immortalized MM cells (211H cells). Interestingly, transcriptional down-regulation of inhibition of differentiation-1 (Id-1) was detected in REIC/Dkk-3-overexpressed 211H cells. Moreover, restoration of Id-1 expression antagonized REIC/Dkk-3-induced JNK phosphorylation and apoptosis. Mutagenesis experiments with the 2.1-kb human Id-1 promoter revealed that activating transcription factor 3 (ATF3) and Smad interaction, with their respective binding motifs, was essential for REIC/Dkk-3-mediated suppression of Id-1 promoter activity. ATF3 activation was probably induced by endoplasmic reticulum stress. Finally, we showed strong antitumor effects from REIC/Dkk-3 gene transfer into the pleural cavity in an orthotopic MM mouse model. Relative to control tumor tissue, REIC/Dkk-3-treated tumor tissue showed down-regulated expression of Id-1 mRNA, enhanced expression of phosphorylated JNK, and an increased number of apoptotic cells. In summary, we first showed that both ATF3 and Smad were crucially and synergistically involved in down-regulation of Id-1, which regulated JNK phosphorylation in REIC/Dkk-3-induced apoptosis. Thus, gene therapy with REIC/Dkk-3 may be a promising therapeutic tool for MM. [Cancer Res 2008;68(20):8333-41]

DOI： 10.1158/0008-5472.CAN-08-0080

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REIC/Dkk-3遺伝子治療の正常細胞を介する抗腫瘍効果(Transduction of gene therapeutic REIC/Dkk-3 into normal cells provides another arm for tumor suppression in vivo)

片岡 健, 阪口 政清, Fernando Abarzua, 谷本 竜太, 渡部 昌実, 村田 等, 那須 保友, 公文 裕巳, 許 南浩

日本癌学会総会記事   67回   93 - 93   2008.9

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REIC/Dkk-3遺伝子によるアポトーシス耐性メカニズムとその克服(BiP is a determining protein for sensitivity to apoptosis induced by a gene-therapeutic adenovirus carrying REIC/Dkk-3)

谷本 竜太, 阪口 政清, アバルズア・フェルナンド, 片岡 健, 那須 保友, 公文 裕巳, 許 南浩

日本癌学会総会記事   67回   238 - 238   2008.9

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Language：English   Publisher：日本癌学会  

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REIC/Dkk-3の強制発現はERストレスを介してIL-7を誘導する(Over-expression of REIC/Dkk-3 induces IL-7 in normal human fibroblasts via ER stress)

阪口 政清, 片岡 健, アバルスア・フェルナンド, 谷本 竜太, 渡辺 昌実, 村田 等, 那須 保友, 公文 裕己, 許 南浩

日本癌学会総会記事   67回   238 - 238   2008.9

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BRPK/PINK1はAktのリン酸化によってがん細胞のアポトーシスを抑制する(BRPK/PINK1 blocks apoptotic cell death of cancer cells by phosphotylation of Akt)

村田 等, 阪口 政清, 片岡 健, 許 南浩

日本癌学会総会記事   67回   64 - 64   2008.9

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S100A8/A9, a key mediator for positive feedback growth stimulation of normal human keratinocytes

Takamasa Nukui, Ritsuko Ehama, Masakiyo Sakaguchi, Hiroyuki Sonegawa, Chika Katagiri, Toshihiko Hibino, Nam-Ho Huh

JOURNAL OF CELLULAR BIOCHEMISTRY   104 ( 2 )   453 - 464   2008.5

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Language：English   Publisher：WILEY-LISS  

S100A8 and S100A9 are known to be up-regulated in hyperproliferative and psoriatic epidermis, but their function in epidermal keratinocytes remains largely unknown. Here we show that (1) S100A8 and S100A9 are secreted by cultured normal human keratinocytes (NHK) in a cytokine-dependent manner, (2) when applied to NHK, recombinant S100A8/A9 (a 1:1 mixture of S100A8 and S100A9) induced expression of a number of cytokine genes such as IL-8/CXCL8, CXCL1, CXCL2, CXCL3, CCL20, IL-6, and TNF alpha that are known to be up-regulated in psoriatic epidermis, (3)the S100A8/ A9-induced cytokines in turn enhanced production and secretion of S100A8 and S100A9 by NHK, and (4) S100A8 and S100A8/A9 stimulated the growth of NHK at a concentration as low as 1 ng/ml. These results indicate the presence of a positive feedback loop for growth stimulation involving S100A8/A9 and cytokines in human epidermal keratinocytes, implicating the relevance of the positive feedback loop to the etiology of hyperproliferative skin diseases, including psoriasis.

DOI： 10.1002/jcb.21639

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S100A11 functions as a dual mediator for growth regulation in normal human keratinocytes

M. Sakaguchi, H. Murata, N. Huh

JOURNAL OF INVESTIGATIVE DERMATOLOGY   128   S138 - S138   2008.4

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：NATURE PUBLISHING GROUP  

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Serum calcium-binding S100A8/A9 proteins in patients with psoriatic arthritis are derived from not only the skin lesions but also circulating monocytes

S. Aochi, K. Tsuji, M. Sakaguchi, N. Huh, T. Tsuda, K. Yamanishi, M. Komine, K. Iwatsuki

JOURNAL OF INVESTIGATIVE DERMATOLOGY   128   S17 - S17   2008.4

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：NATURE PUBLISHING GROUP  

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S100A11, an dual mediator for growth regulation of human keratinocytes

Masakiyo Sakaguchi, Hiroyuki Sonegawa, Hitoshi Murata, Midori Kitazoe, Jun-ichiro Futami, Ken Kataoka, Hidenori Yamada, Nam-ho Huh

MOLECULAR BIOLOGY OF THE CELL   19 ( 1 )   78 - 85   2008.1

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Language：English   Publisher：AMER SOC CELL BIOLOGY  

We previously revealed a novel signal pathway involving S100A11 for inhibition of the growth of normal human keratinocytes (NHK) caused by high Ca++ or transforming growth factor beta. Exposure to either agent resulted in transfer of S100A11 to nuclei, where it induced p21(WAF1). In contrast, S100A11 has been shown to be overexpressed in many human cancers. To address this apparent discrepancy, we analyzed possible new functions of S100A11, and we provide herein evidence that 1) S100A11 is actively secreted by NHK; 2) extracellular S100A11 acts on NHK to enhance the production of epidermal growth factor family proteins, resulting in growth stimulation; 3) receptor for advanced glycation end products, nuclear factor-kappa B, Akt, and cAMP response element-binding protein are involved in the S100A11-triggered signal transduction; and 4) production and secretion of S100A11 are markedly enhanced in human squamous cancer cells. These findings indicate that S100A11 plays a dual role in growth regulation of epithelial cells.

DOI： 10.1091/mbc.E07-07-0682

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Truncation of annexin A1 is a regulatory lever for linking epidermal growth factor signaling with cytosolic phospholipase A2 in normal and malignant squamous epithelial cells

Masakiyo Sakaguchi, Hitoshi Murata, Hiroyuki Sonegawa, Yoshihiko Sakaguchi, Jun-ichiro Futami, Midori Kitazoe, Hidenori Yamada, Nam-ho Huh

JOURNAL OF BIOLOGICAL CHEMISTRY   282 ( 49 )   35679 - 35686   2007.12

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Regulation of cell growth and apoptosis is one of the pleiotropic functions of annexin A1 (ANXA1). Although previous reports on the overexpression of ANXA1 in many human cancers and on growth suppression and/or induction of apoptosis by ANXA1 may indicate the tumor-suppressive nature of ANXA1, molecular mechanisms of the function of ANXA1 remain largely unknown. Here we provide evidence that ANXA1 mechanistically links the epidermal growth factor-triggered growth signal pathway with cytosolic phospholipase A(2) (cPLA(2)), an initiator enzyme of the arachidonic acid cascade, through interaction with S100A11 in normal human keratinocytes (NHK). Ca2+ -dependent binding of S100A11 to ANXA1 facilitated the binding of the latter to cPLA2, resulting in inhibition of cPLA2 activity, which is essential for the growth of NHK. On exposure of NHK to epidermal growth factor, ANXA1 was cleaved solely at Trp(12), and this cleavage was executed by cathepsin D. In squamous cancer cells, this pathway was shown to be constitutively activated. The newly found mechanistic intersection may be a promising target for establishing new measures against human cancer and other cell growth disorders.

DOI： 10.1074/jbc.M707538200

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Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure

Masahiro Miyazaki, Marhaen Hardjo, Takuro Masaka, Koji Tomiyama, Naila Mahmut, Reinhold J. Medina, Aya Niida, Hiroyuki Sonegawa, Gang Du, Rong Yong, Mikiro Takaishi, Masakiyo Sakaguchi, Nam-Ho Huh

STEM CELLS   25 ( 11 )   2855 - 2863   2007.11

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Language：English   Publisher：ALPHAMED PRESS  

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, similar to 24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1 alpha, and HNF4 alpha. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases.

DOI： 10.1634/stemcells.2007-0078

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ヒト膀胱癌細胞におけるREIC/Dkk-3誘導性アポトーシスに対する抵抗性の機構解析(Mechanistic Analysis of Resistance to REIC/Dkk-3-induced Apoptosis in Human Bladder Cancer Cell Lines)

小林 知子, 阪口 政清, 谷本 竜太, 枝村 康平, アバラズア・フェルナンド, 片岡 健, 賀来 春紀, 雑賀 隆史, 宮崎 正博, 那須 保友, 公文 裕巳, 許 南浩

西日本泌尿器科   69 ( 増刊 )   125 - 125   2007.10

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Language：English   Publisher：西日本泌尿器科学会  

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Adenovirus-mediated REIC/Dkk-3 gene transfer inhibits tumor growth and metastasis in an orthotopic prostate cancer model

K. Edamura, Y. Nasu, M. Takaishi, T. Kobayashi, F. Abarzua, M. Sakaguchi, Y. Kashiwakura, S. Ebara, T. Saika, M. Watanabe, N-H Huh, H. Kumon

CANCER GENE THERAPY   14 ( 9 )   765 - 772   2007.9

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We had previously reported that REIC/Dkk-3, a member of the Dickkopf ( Dkk) gene family, works as a tumor suppressor. In this study, we evaluated the therapeutic effects of an intratumoral injection with adenoviral vector encoding REIC/Dkk-3 gene ( Ad-REIC) using an orthotopic mouse prostate cancer model of RM-9 cells. We also investigated the in vivo anti-metastatic effect and in vitro anti-invasion effect of Ad-REIC gene delivery. We demonstrated that the Ad-REIC treatment inhibited prostate cancer growth and lymph node metastasis, and prolonged mice survival in the model. These therapeutic responses were consistent with the intratumoral apoptosis induction and in vitro suppression of cell invasion/migration with reduced matrix metalloprotease-2 activity. We thus concluded that in situ Ad-REIC/Dkk-3 gene transfer may be a promising therapeutic intervention modality for the treatment of prostate cancer.

DOI： 10.1038/sj.cgt.7701071

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ヒト膀胱癌細胞株におけるREIC/Dkk-3誘導性アポトーシスに対する抵抗性の機構的解析(Mechanistic Analysis of Resistance to REIC/Dkk-3-induced Apoptosis in Human Bladder Cancer Cell Lines)

小林 知子, 阪口 政清, 谷本 竜太, アバラズア・フェルナンド, 高石 樹朗, 片岡 健, 賀来 春紀, 雑賀 隆史, 那須 保友, 宮崎 正博, 公文 裕巳, 許 南浩

日本癌学会総会記事   66回   476 - 476   2007.8

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Heat shock proteins play a crucial role in tumor-specific apoptosis by REIC/Dkk-3

Fernando Abarzua, Masakiyo Sakaguchi, Ryuta Tanimoto, Hiroyuki Sonegawa, Dai-Wei L, Kohei Edamura, Tomoko Kobayashi, Masami Watanabe, Yuji Kashiwakura, Haruki Kaku, Takashi Saika, Keiichiro Nakamura, Yasutomo Nasu, Hiromi Kumon, Nam-Ho Huh

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE   20 ( 1 )   37 - 43   2007.7

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We recently showed that overexpression of REIC/Dickkopf-3 (Dkk-3), a tumor suppressor gene, induced apoptosis in a tumor cell-specific manner. The aim of the present study was to determine the mechanisms underlying the selective induction of apoptosis. At first, we found a mouse renal carcinoma cell line, RENCA, to be extremely sensitive to an adenovirus carrying REIC/Dkk-3 (Ad-REIC), and we showed that activation of c-Jun N-terminal kinase (JNK) was a critical step in cell death, i.e. a process similar to that in human prostate and testicular cancer observed in our previous studies. Among the proteins interfering with the activation of JNK, heat shock protein (Hsp)70/72 was reduced in expression in RENCA cells compared with that in NIH3T3 cells. An Hsp70/72 inducer protected RENCA cells from Ad-REIC-induced apoptosis, while an Hsp70/72 inhibitor sensitized NIH3T3 cells for apoptosis induction. These results indicate that functionally active Hsp70/72 is a key factor in tumor cell-specific induction of apoptotic cell death and that analyses of the expression levels of Hsp70/72 may be essential in determining the significance of Ad-REIC-based gene therapy against human cancer.

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Involvement of deterioration in S100C/A11-mediated pathway in resistance of human squamous cancer cell lines to TGF beta-induced growth suppression

Hiroyuki Sonegawa, Takamasa Nukui, Dai-Wei Li, Mikiro Takaishi, Masakiyo Sakaguchi, Nam-Ho Hub

JOURNAL OF MOLECULAR MEDICINE-JMM   85 ( 7 )   753 - 762   2007.7

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Recently, we demonstrated that S100C/A11 comprises an essential pathway for growth suppression by TGF beta in normal human keratinocytes. Nuclear transfer of S 100C/A11 was a hallmark of the activation of the process. In the present study, we examined the possible deterioration in the pathway in human squamous cancer cell lines, focusing on intracellular localization of S100C/A11 and its functional partners Smad3 and Smad4. All four human squamous cancer cell lines examined (A431, BSCC-93, DJM-1, and HSC-5) were resistant to growth suppression by TGF beta. In BSCC-93, DJM-1, and HSC-5 cells exposed to TGF beta, S100C/A11 was not transferred to the nuclei, and p21(WAF1) was not induced. Overexpression of nucleus-targeted S100C/A11 partially recovered induction of p21 (WAF1) and p15(INK4B) and growth suppression by TGF beta 1 in these cells. These results indicate that the deterioration in the S100C/A11-mediated pathway conferred upon the cancer cell lines resistance to TGF beta. In A431 cells, S100C/A11, Smad3, and Smad4 were simultaneously transferred to the nuclei, and p2l(WAF1) was induced upon exposure to TGF beta. We provide evidence to indicate that refractoriness of A431 cells to TGF beta was probably because the amount of p2l(WAF1) induced by TGF beta was insufficient to counteract cyclin A, which is highly overexpressed in A431 cells. Thus, the newly found S100C/A11-mediated pathway is at least partly involved in conferring upon human squamous cell cancers resistant to TGF beta-induced growth suppression, which is considered to play a critical role for the initiation and progression of many human cancers.

DOI： 10.1007/s00109-007-0180-7

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S100A8/A9, a key mediator for positive feedback growth stimulation and inflammation of normal human keratinocytes

R. Ehama, T. Nukui, C. Katagiri, M. Sakaguchi, H. Sonegawa, N. Huh, T. Hibino

JOURNAL OF INVESTIGATIVE DERMATOLOGY   127   S98 - S98   2007.4

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Involvement of deterioration in S100C/A11-mediated pathway in resistance of human squamous cancer cell lines to TGF beta-induced growth suppression

SONEGAWA HIROYUKI, SAKAGUCHI MASAKIYO, HUH NAM-HO

26 ( 1 )   63 - 63   2007.3

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S100A8/A9, a Key Mediator for Positive Feedback Growth Stimulation of Normal Human Keratinocytes

EHMA RITSUKO, NUKUI TAKAMASA, SAKAGUCHI MASAKIYO, SONEGAWA HIROYUKI, KATAGIRI CHIKA, HIBINO TOSHIHIKO, HUH NAM-HO

26 ( 1 )   73 - 73   2007.3

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Autocrine growth-stimulation of normal human keratinocytes by secreted S100C/A11

SAKAGUCHI MASAKIYO, SONEGAWA HIROYUKI, HUH NUM-HO

26 ( 1 )   74 - 74   2007.3

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REIC/Dkk-3 as a potential gene therapeutic agent against human testicular cancer

TANIMOTO RYUTA, SAKAGUCHI MASAKIYO, FERNANDO ABARZUA, SONEGAWA HIROYUKI, NASU YASUTOMO, KUMON HIROMI, HUH NUM-HO

26 ( 1 )   88 - 88   2007.3

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REIC/Dkk-3 as a potential gene therapeutic agent against human testicular cancer

Ryuta Tanimoto, Fernando Abarzua, Masakiyo Sakaguchi, Mikiro Takaishi, Yasutomo Nasu, Hiromi Kumon, Nam-Ho Huh

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE   19 ( 3 )   363 - 368   2007.3

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Human testicular cancer is very sensitive to chemotherapy and radiation therapy and is regarded as a curable cancer. The cancer prevails in the young reproductive generation and testicular dysfunction is often observed as a side effect, remaining a serious challenge. In the present study, we examined the potential utility of REIC/Dkk-3-based gene therapy against human testicular cancer. Expression of REIC/Dkk-3 was reduced in all of the human seminoma and non-seminomatous germ cell tumor tissues. Overexpression of REIC/Dkk-3 using an adenovirus vector (Ad-REIC) induced apoptosis in a testicular germ cell cancer cell line NCCIT but not in normal human fibroblasts. c-Jun terminal kinase (JNK) was activated by Ad-REIC and the induction of apoptosis was abrogated by a JNK inhibitor. A single intratumoral injection of Ad-REIC markedly inhibited the tumorigenic growth of NCCIT cells in nude mice. These results indicate that Ad-REIC may lead to developing less insulting and non-genotoxic therapeutic measures against human testicular cancer.

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Denatured and reversibly cationized p53 readily enters cells and simultaneously folds to the functional protein in the cells

H Murata, M Sakaguchi, J Futami, M Kitazoe, T Maeda, H Doura, M Kosaka, H Tada, M Seno, N Huh, H Yamada

BIOCHEMISTRY   45 ( 19 )   6124 - 6132   2006.5

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Cationization is a powerful strategy for internalizing a protein into living cells. On the other hand, a reversibly cationized denatured protein through disulfide bonds is not only soluble in water but also able to fold to the native conformation in vitro. When these advantages in cationization were combined, we developed a novel method to deliver a denatured protein into cells and simultaneously let it fold to express its function within cells. This "in-cell folding" method enhances the utility of recombinant proteins expressed in Escherichia coli as inclusion bodies; that is, the recombinant proteins in inclusion bodies are solubilized by reversible cationization through cysteine residues by disulfide bonds with aminopropyl methanethiosulfonate or pyridyldithiopropionylpolyethylenimine and then incubated with cells without an in vitro folding procedure. As a model protein, we investigated human tumor-suppressor p53. Treatment of p53-null Saos-2 cells with reversibly cationized p53 revealed that all events examined as indications of the activation of p53 in cells, such as reduction of disulfide bonds followed by tetramer formation, localization into the nucleus, induction of p53 target genes, and induction of apoptosis of cells, occurred. These results suggest that reversible cationization of a denatured protein through cysteine residues is an alternative method for delivery of a functional protein into cells. This method would be very useful when a native folded protein is not readily available.

DOI： 10.1021/bi052642a

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Rescue of 90%-hepatectomized rats with acute liver failure by intrasplenic transplantation of hepatocytes derived from rat bone marrow cells

MIYAZAKI MASAHIRO, HARDJO MARHAEN, MEDINA REINHOLD, MASAKA TAKURO, NAILA MAHMUT, TOMIYAMA KOJI, SAKAGUCHI MASAKIYO, TAKAHASHI MIKIRO, HUH NAM-HO

25 ( 1 )   75 - 75   2006.3

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In vitro trans-differentiation of bone marrow derived hepatocyte-like cells into insulin producing cells

MASAKA TAKURO, MIYAZAKI MASAHIRO, HARDJO MARHAEN, MEDINA REINHOLD, SAKAGUCHI MASAKIYO, TAKAISHI MIKIRO, HUH NAM-HO

25 ( 1 )   77 - 77   2006.3

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HNF4 転写因子による肝機能制御機構の解明

阪口政清

ファルマシア   2006

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Adenovirus-mediated overexpression of REIC/Dkk-3 selectively induces apoptosis in human prostate cancer cells through activation of c-Jun-NH2-kinase

F Abarzua, M Sakaguchi, M Takaishi, Y Nasu, K Kurose, S Ebara, M Miyazaki, M Namba, H Kumon, N Huh

CANCER RESEARCH   65 ( 21 )   9617 - 9622   2005.11

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Alteration in genes which takes place during malignant conversion and progression could be potential targets for gene therapy. We previously identified REIC/Dkk-3 as a gene whose expression is reduced in many human cancers. Here, we showed that expression of REIC/Dkk-3 was consistently reduced in human prostate cancer tissues in a stage-dependent manner. Forced expression of REIC/Dkk-3 induced apoptosis in human prostate cancer cell lines lacking endogenous REIC/Dkk-3 expression but not in REIC/Dkk3-proficient normal prostate epithelial and stromal cells. The apoptosis involved c-jun-NH2-kinase activation, mitochondrial translocation of Bax, and reduction of Bcl-2. A single injection of an adenovirus vector carrying REIC/Dkk-3 showed a dramatic antitumor effect on a xenotransplanted human prostate cancer. Thus, REIC/Dkk-3 could be a novel target for gene-based therapy of prostate cancer.

DOI： 10.1158/0008-5472.CAN-05-0829

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Bifurcated converging pathways for high Ca2+- and TGF beta-induced inhibition of growth of normal human keratinocytes

M Sakaguchi, H Sonegawa, T Nukui, Y Sakaguchi, M Miyazaki, M Namba, N Huh

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   102 ( 39 )   13921 - 13926   2005.9

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Growth suppression of normal human keratinocytes by high Ca2+ or TGF beta was shown to be mediated by p2l(WAF1/C1P1) and Sp1 [Pardali, K., et al. (2000) J. Biol. Chem. 275, 29244-29256; Santini, M. P., Talora, C., Seki, T., Bolgan, L. & Dotto, G. P. (2001) Proc. Nat. Acad Sci. USA 98, 9575-9580; Al-Daraji, W. I., Grant, K. R., Ryan, K., Saxton, A., & Reynolds, N. J. (2002) J. Invest. Dermato/. 118, 779-788]. We previously demonstrated that S100C/A11 is a key mediator for growth inhibition of normal human epidermal keratinocytes (NHK) triggered by high Ca2+ or TGF beta [Sakaguchi, M., et al. (2003) J. Cell Biol. 163, 825-835; Sakaguchi, M., et al. (2004) 164, 979-984]. On exposure of NHK cells to either agent, S100C/ All is transferred to nuclei, where it induces p21(WAF1/CIP1) through activation of Sp1/Sp3. In the present study, we found that high Ca2+ activated NFAT1 through calcineurin-dependent dephosphorylation. In growing NHK cells, Krueppel-like factor (KLF)16, a member of the Sp/KLF family, bound to the p21(WAF1/CIP1) promoter and, thereby, inhibited the transcription of p21(WAF1/CIP1). Sp1 complexed with NFAT1 in high Ca2+ -treated cells or with Smad3 in TGF beta 1-treated cells, but not Sp1 alone, replaced KLF16 from the p21(WAF1/CIP1) promoter and transcriptionally activated the p21(WAF1/CIP1) gene. Thus, high Ca2+ and TGF beta 1 have a common S1OOC/A11-mediated pathway in addition to a unique pathway (NFAT1-mediated pathway for high Ca2+ and Smad-mediated pathway for TGF beta 1) for exhibiting a growth inhibitory effect on NHK cells, and both pathways were shown to be indispensable for growth inhibition.

DOI： 10.1073/pnas.0500630102

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Protein transduction assisted by polyethylenimine-cationized carrier proteins

M Kitazoe, H Murata, J Futami, T Maeda, M Sakaguchi, M Miyazaki, M Kosaka, H Tada, M Seno, N Huh, M Namba, M Nishikawa, Y Maeda, H Yamada

JOURNAL OF BIOCHEMISTRY   137 ( 6 )   693 - 701   2005.6

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Previously, we have reported that cationized-proteins covalently modified with polyethylenimine (PEI) (direct PEI-cationization) efficiently enter cells and function in the cytosol [Futami et al. (2005) J. Biosci. Bioeng. 99,95-1031. However, it may be more convenient if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a specific affinity (indirect PEI-cationization). Thus, we prepared PEI-cationized avidin (PEI-avidin), streptavidin (PEI-streptavidin), and protein G (PEI-protein G), and examined whether they could deliver biotinylated proteins and antibodies into living cells. PEI-avidin (and/or PEI-streptavidin) carried biotinylated GFPs into various mammalian cells very efficiently. A GFP variant containing a nuclear localization signal was found to arrive even in the nucleus. The addition of a biotinylated RNase A derivative mixed with PEI-streptavidin to a culture medium of 3T3-SV-40 cells resulted in remarkable cell growth inhibition, suggesting that the biotinylated RNase A derivative entered cells and digested intracellular RNA molecules. Furthermore, the addition of a fluorescein-labeled antiS100C (beta-actin binding protein) antibody mixed with PEI-protein G to human fibroblasts resulted in the appearance of a fluorescence image of actin-like filamentous structures in the cells. These results indicate that indirect PEI-cationization using non-covalent interaction is as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their functions in the cytosol. Thus, PEI-cationized proteins having a specific affinity for certain molecules such as PEI-streptavidin, PEI-avidin and PEI-protein G are concluded to be widely applicable protein transduction carrier molecules.

DOI： 10.1093/jb/mvi081

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Intracellular delivery of proteins into mammalian living cells by polyethylenimine-cationization

J Futami, M Kitazoe, T Maeda, E Nukui, M Sakaguchi, J Kosaka, M Miyazaki, M Kosaka, H Tada, M Seno, Y Sasaki, NH Huh, M Namba, H Yamada

JOURNAL OF BIOSCIENCE AND BIOENGINEERING   99 ( 2 )   95 - 103   2005.2

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In the post-genomic era, there is pressing need for development of protein manipulation methodology to analyze functions of proteins in living cells. For this purpose, techniques to deliver functional proteins into living cells are currently being evaluated as alternative approaches to the introduction of transcriptionally active DNA. Here, we describe a novel method for efficient protein transduction into living cells in which a protein is simply cationized with polyethylenimine (PEI) by limited chemical conjugation. PEI-cationized proteins appear to adhere to the cell surface by ionic charge interaction and then internalize into cells in a receptor- and transporter-independent fashion. Since PEI is an organic macromolecule with a high cationic-charge density, limited coupling with PEI results in endowment of sufficient cationic charge to proteins without causing serious decline in their fundamental functions. A number of PEI-cationized proteins, such as ribonuclease (RNase), green fluorescent protein (GFP) and immunoglobulin (IgG), efficiently entered cells and functioned in the cytosol. Our results suggest that protein cationization techniques using PEI will be useful for the development of protein transduction technology.

DOI： 10.1263/jbb.99.095

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Targeted disruption of transcriptional regulatory function of p53 by a novel efficient method for introducing a decoy oligonucleotide into nuclei

M Sakaguchi, T Nukui, H Sonegawa, H Murata, J Futami, H Yamada, N Huh

NUCLEIC ACIDS RESEARCH   33 ( 9 )   2005

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Decoy oligonucleotides have been used for functional sequestering of transcription factors. Efficient introduction into cells is a prerequisite for the oligonucleotides to exert their blocking function. Lipofection is the most widely used technique for that purpose because of its convenience and relatively high efficiency. However, the transduction efficiency of lipofection largely depends on cell types and experimental conditions and the introduced nucleotides are not specifically directed to nuclei where they exert their major function. In the present study, we designed a new system for transporting oligonucleotides into cell nuclei. The vehicle is composed of glutathione-S-transferase, 7 arginine residues, the DNA- binding domain of GAL4 and a nuclear localization signal, which are linked with flexible glycine stretches. The p53-responsive element linked to the GAL4 upstream activating sequence was efficiently transferred by the vehicle protein into nuclei of primary cultures of neuronal cells, embryonic stem cells and various human normal cells. Transcriptional activation of p21(WAF1/CIP1) and Bax by p53 on exposure to cisplatin was completely blocked by introducing the p53 decoy oligonucleotide. Thus, the system developed in the present study can be a convenient and powerful tool for specifically disrupting the function of DNA- binding proteins in culture.

DOI： 10.1093/nar/gni088

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siRNAによる遺伝子発現抑制法

阪口政清, 曽根川裕之, 温井孝昌, 許 南浩

Surgery Frontier   2005

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PKCa mediates TGFb-induced growth inhibition of human keratinocytes via phosphorylation of S100C/A11

M Sakaguchi, M Miyazaki, H Sonegawa, M Kashiwagi, M Ohba, T Kuroki, M Namba, N Huh

MOLECULAR BIOLOGY OF THE CELL   15   449A - 449A   2004.11

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CELL BIOLOGY  

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Increased expression of calcium-binding protein S100 in human uterine smooth muscle tumours

T Kanamori, K Takakura, M Mandai, M Kariya, K Fukuhara, M Sakaguchi, NH Huh, K Saito, T Sakurai, J Fujita, S Fujii

MOLECULAR HUMAN REPRODUCTION   10 ( 10 )   735 - 742   2004.10

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S100 proteins belong to the EF-hand Ca2+ -binding protein family and regulate a variety of cellular processes via interaction with different target proteins. Several diseases, including cancer and melanoma, are related to the abnormal expression of S100 proteins, which are expressed in cell- and tissue-specific manners. We investigated the expression of S100 family members in human uterine smooth muscle tumours. Expression of six members of the S100 protein family: S100A1, A4, A6, A7, A10 and A11, was found in human uterine leiomyoma and myometrium tissue, but expression of other members was not detected by RT-PCR. Real-time PCR showed that S100A11 expression was significantly increased in leiomyoma compared with myometrium. Suppression of S100A11 by small interfering RNA (siRNA) led to apoptosis, and the overexpression of S100A11 inhibited apoptosis in human uterine smooth muscle tumour cells. These findings suggest that S100A11 has an anti-apoptotic function and is related to the process of growth of human uterine leiomyoma.

DOI： 10.1093/molehr/gah100

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Expression of CYP3A4 by an immortalized human hepatocyte line in a three-dimensional culture using a radial-flow bioreactor

Akiyama, I, K Tomiyama, M Sakaguchi, M Takaishi, M Mori, M Hosokawa, S Nagamori, N Shimizu, NH Huh, M Miyazaki

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE   14 ( 4 )   663 - 668   2004.10

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Cytochrome P450 (CYP) 3A is responsible for about 50% of drug metabolizing activity in the liver. The present study was undertaken to establish a CYP3A4-active model for in vitro analysis of human drug metabolism. The cells used were immortalized normal human fetal hepatocytes (OUMS-29) and its HNF4alpha-introduced subline (OUMS-29/H-11). The cells were cultivated under high-density three-dimensional conditions in a radial-flow bioreactor (RFB). The number of OUMS-29 cells increased 15-fold over 49 days and their apical surfaces were covered with abundant microvilli, a characteristic of hepatocytes in vivo. The amount of albumin secreted by OUMS-29 cells in the three-dimensional RFB culture was 6-fold higher than those in a monolayer culture. CYP3A4 protein and an intermediate metabolite of testosterone by CYP3A4 were detected in OUMS-29/H11 cells cultivated in RFB >29 days. These results indicate that the RFB; culture of OUMS-29/H-11 cells is useful for screening and developing new drugs.

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Introduction of an N-terminal peptide of S100C/A11 into human cells induces apoptotic cell death

E Makino, M Sakaguchi, K Iwatsuki, N Huh

JOURNAL OF MOLECULAR MEDICINE-JMM   82 ( 9 )   612 - 620   2004.9

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S100 proteins belong to the EF-hand Ca2+- binding protein family and are involved in the regulation of a variety of cellular processes. Individual S 100 proteins are expressed in cell- and tissue-specific manners, and functional deterioration of S100 proteins leads to a number of human diseases, including cancer. We previously demonstrated that S100C/A11 was translocated to nuclei and inhibited DNA synthesis in human keratinocytes when exposed to high Ca2+. In the present study we examined the effects of synthetic partial peptides of S100C/ A11 on human carcinoma cell lines. Only an N-terminal peptide with 19 amino acid residues (MAK19) showed cytotoxicity to the cell lines in dose- and time-dependent manners when introduced into cells by flanking the HIV-TAT protein transduction domain (TAT-MAK19). Pulse field electrophoresis revealed that DNA of the treated cells was partially degradated. Annexin V, a marker of cellular apoptosis, was detected in the cells treated with TAT-MAK19 by immunostaining and flow cytometry. The induction of apoptotic cell death was apparently independent of p53, p21(WAF1/CIP1), and caspase activity, but treatment with TAT-MAK19 resulted in partial translocation of apoptosis-inducing factor (AIF) from the cytoplasm to nuclei. These results indicate that MAK19 induces apoptosis in human cell lines and may therefore lead to the establishment of a new molecular target for the treatment of human cancer.

DOI： 10.1007/s00109-004-0560-1

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Involvement of interferon regulatory factor 1 and S100C/A11 in growth inhibition by transforming growth factor beta 1 in human hepatocellular carcinoma cells

M Miyazaki, M Sakaguchi, Akiyama, I, Y Sakaguchi, S Nagamori, NH Huh

CANCER RESEARCH   64 ( 12 )   4155 - 4161   2004.6

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Growth inhibition by transforming growth factor (TGF)-beta1 has been attributed to the induction of cyclin-dependent kinase inhibitors, among which p21/Waf1 plays a major role in many biological contexts. In the present study, two new intracellular mediators for the induction of p21/Waf1 by TGF-beta1 were identified in a human hepatocellular carcinoma cell line (JHH-5) expressing mutant-type p53. After addition of TGF-beta1 to JHH-5 cells, a marked increase of the p21/Waf1 expression preceded the inhibition of DNA synthesis. Expression of IFN regulatory factor (IRF)-1, a known transacting factor for p21/Waf1 promoter, was elevated just before or in parallel with the increase of p21/Waf1. Transduction of antisense IRF-1 inhibited the increase in p21/Waf1 in JHH-5 cells treated with TGF-beta1 and partially released the cells from the growth arrest by TGF-beta1. Expression of S100C/A11, a member of the Ca2+-binding S100 protein family, also markedly increased after addition of TGF-beta1. S100C/A11 protein was translocated to and accumulated in nuclei of TGF-beta1-treated JHH-5 cells, where p21/Waf1 was concomitantly accumulated. When a recombinant S100C/A11 protein was introduced into nuclei of JHH-5 cells, DNA synthesis was markedly inhibited in a dose-dependent manner in the absence of TGF-beta1. Prior transfection of p21/Waf1-targeted small interfering RNA efficiently blocked decrease of DNA synthesis in JHH-5 cells caused by TAT-S100C/A11 or TGF-beta1 and markedly inhibited expression of p21/Waf1 protein in the cells. These results indicate that IRF-1 and S100C/A11 mediate growth inhibition by TGF-beta1 via induction of p21/Waf1.

DOI： 10.1158/0008-5472.CAN-03-2750

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Establishment of an immortalized human-liver endothelial cell line with SV40T and hTERT

T Matsumura, M Takesue, KA Westerman, T Okitsu, M Sakaguchi, T Fukazawa, T Totsugawa, H Noguchi, S Yamamoto, DB Stolz, N Tanaka, P Leboulch, N Kobayashi

TRANSPLANTATION   77 ( 9 )   1357 - 1365   2004.5

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Background and Aims. Liver endothelial cells (LECs) perform an essential role in important pathophysiologic functions in the liver. Establishment of a human LEC line facilitates advances in LEC research. Here, we present immortalization of human LECs using retroviral gene transfer of simian virus 40 large T antigen (SV40T) and human telomerase reverse transcriptase (hTERT). We also demonstrate excision of SV40T and hTERT with TAT-mediated Cre/loxP recombination and subsequent cell sorting.
Methods. First, human LECs were transduced with a retroviral vector somatostatin receptor (SSR)#69 expressing SV40T and hygromycin-resistance genes flanked by a pair of loxA recombination targets. Then, cells were retrovirally superinfected with SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs that were intervened by two loxBs. One SV40T- and hTERT-immortalized LEC clone, TMNK-1, was established and analyzed for its biologic characteristics.
Results. The cells were hygromycin-resistant and uniformly positive for GFP expression. TMNK-1 expressed EC markers, including factor VIII, vascular endothelial growth factor receptors (flt-1, KDR/Flk-1), and CD34, showed uptake of Di-I-acetylated-low-density lipoprotein and angiogenic potential in Matrigel assays. After lipopolysaccharide treatment, TMNK-1 produced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 and exhibited increased expression of intracellular adhesive molecule-1, vascular cellular adhesive molecule-1, and VE-cadherin. After treatment with TAT-Cre recombinase fusion protein, approximately 60% of TMNK-1 was negative for GFP expression, and subsequent cell sorting of this population for GFP allowed for collection of the reverted form of TMNK-1.
Conclusions. This study demonstrates the utility and efficiency of the reversible immortalization procedure to expand primary human LECs for basic studies.

DOI： 10.1097/01.TP.0000124286.82961.7E

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Differential expression of S100C in thyroid lesions

C Torres-Cabala, A Panizo-Santos, HC Krutzsch, H Barazi, M Namba, M Sakaguchi, DD Roberts, MJ Merino

INTERNATIONAL JOURNAL OF SURGICAL PATHOLOGY   12 ( 2 )   107 - 115   2004.4

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Language：English   Publisher：WESTMINSTER PUBL INC  

Identification of new potential markers that may help in the diagnosis of benign and malignant thyroid lesions is needed. By comparative 2-dimensional gel electrophoresis of microdissected cells from tumors and normal thyroid tissue, we identified a new protein, S100C, which is highly expressed in papillary carcinomas. In order to validate this finding, we investigated the immunohistochemical expression and the potential role in diagnosis of these markets in 94 specimens representing the spectrum of malignant and benign thyroid lesions. Normal thyroid tissue was evaluated in 57 specimens. Galectin-3, a marker reported as specific for malignant lesions, was also evaluated in the same lesions. S100C protein was expressed in the nuclei of normal tissue, hyperplastic nodules, and follicular adenomas and carcinomas. Papillary carcinomas showed a strong, but cytoplasmic, pattern of staining. Galectin-3 immunostaining was strongly positive in papillary carcinomas, and negative in benign lesions, confirming its value in differential diagnosis. These findings Suggest that immunohistochemical Staining of S100C could be helpful in the pathological study of thyroid lesions, especially in cases in which follicular variants of papillary carcinoma and follicular carcinoma are considered in the differential diagnosis.

DOI： 10.1177/106689690401200203

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Growth Regulation of Normal Human Keratinocytes : PKCa Mediates TGFb-induced Growth Inhibition via Phosphorylation of S100C/A11

SAKAGUCHI Masakiyo, MIYAZAKI Masahiro, SONEGAWA Hiroyuki, KASHIWAGI Mariko, OHBA Motoi, KUROKI Toshio, NAMBA Masayoshi, HUH Namho

23 ( 1 )   38 - 38   2004.3

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Decreased expression of REIC/Dkk-3 in human renal clear cell carcinoma

K Kurose, M Sakaguchi, Y Nasu, S Ebara, H Kaku, R Kariyama, Y Arao, M Miyazaki, T Tsushima, M Namba, H Kumon, NH Huh

JOURNAL OF UROLOGY   171 ( 3 )   1314 - 1318   2004.3

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Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

Purpose: We examined the expression of REIC/Dkk-3, a possible candidate for a tumor suppressor gene, in human renal clear cell carcinoma (RCCC) cell lines and sporadic RCCC surgical specimens.
Materials and Methods: Human RCCC cell lines (Caki-1, Caki-2, ACHN and KPK-1) and several control cell lines were used to examine the expression of REIC/Dkk-3 mRNA and characterize a newly raised antibody specific for REIC/Dkk-3 protein. Pairs of cancerous and adjacent noncancerous tissues were obtained from 20 patients with RCCC. Of them 17 and 7 cases were analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction, and by Western blot analysis and/or immunohistochemical analysis, respectively.
Results: The decreased expression of REIC/Dkk-3 mRNA and protein in human RCCC cell lines, and the specificity of the new antibody were confirmed. In a real-time quantitative reverse transcriptase-polymerase chain reaction study using 17 pairs of RCCC and adjacent normal tissues REIC/Dkk-3 mRNA levels were significantly decreased in carcinoma tissues (by 25% to approximately 95% in 15 pairs). Western blot analysis and immunohistochemistry revealed a significant decrease in REIC/Dkk-3 protein levels in 6 of the 7 and 13 of the 14 RCCC cases analyzed, respectively.
Conclusions: The decrease in REIC/Dkk-3 mRNA and protein levels was observed irrespective of tumor grade and stage, indicating the involvement of REIC/Dkk-3 in an initial step of malignant conversion. Consequently REIC/Dkk-3 could be a new molecular target for therapeutic measures against RCCC.

DOI： 10.1097/01.ju.0000101047.64379.d4

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Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT

M Maruyama, N Kobayashi, KA Westerman, M Sakaguchi, JE Allain, T Totsugawa, T Okitsu, T Fukazawa, A Weber, DB Stolz, P Leboulch, N Tanaka

TRANSPLANTATION   77 ( 3 )   446 - 451   2004.2

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Background. Cholangiocytes perform an essential role in important pathophysiologic functions in the liver. Establishment of a human cholangiocyte line facilitates advances in cholangiocyte research and clinical applications for cell therapies. Here, we describe the immortalization of human cholangiocytes using serial transfection of simian virus 40 large T (SV40T) followed by human telomerase reverse transcriptase (hTERT).
Methods. SV40T-transduced human liver OUMS-21 cells were superinfected with a retroviral vector SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs. Resulting cell lines were evaluated for gene expression, functional cholangiogenic characteristics in vitro and in vivo, and response to lipopolysaccharide (LPS).
Results. One of the SV40T- and hTERT-immortalized cholangiocyte clones, MMNK-1, was established. MMNK-1 expressed cholangiocyte markers, including cytokeratin (CK)-7 and -19 and exhibited cholangiogenic tubule formation in a Matrigel assay. When transplanted into the immunodeficient mice, MMNK-1 cells developed bile duct-like structures in the spleen. After LPS treatment, MMNK-1 cells produced interleukin-6 and failed to form well-developed tubular structures in Matrigel.
Conclusion. We have established an immortalized cholangiocyte cell line, MMNK-1, using SV40T and hTERT transduction.

DOI： 10.1097/01.TP.0000110292.73873.25

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Transplantation of reversibly immortalized insulin-secreting human hepatocytes controls diabetes in pancreatectomized pigs

T Okitsu, N Kobayashi, HS Jun, S Shin, SJ Kim, J Han, H Kwon, M Sakaguchi, T Totsugawa, M Kohara, KA Westerman, N Tanaka, P Leboulch, JW Yoon

DIABETES   53 ( 1 )   105 - 112   2004.1

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Language：English   Publisher：AMER DIABETES ASSOC  

Type 1 diabetes results from the destruction of insulin-producing pancreatic beta-cells by a beta-cell-specific autoimmune process. Although converting other cell types into insulin-producing cells may compensate for the loss of the beta-cell mass while evading beta-cell-specific T-cell responses, proof-of-principle of this approach in large animal models is lacking. This investigation was initiated to determine whether an insulin-producing human hepatocyte line can control diabetes when transplanted into totally pancreatectomized diabetic pigs. We established a reversibly immortalized human hepatocyte line, YOCK-13, by transferring a human telomerase reverse transcriptase cDNA and a drug-inducible Cre recombinase cassette, followed by cDNA for a modified insulin under the control of the L-type pyruvate kinase (L-PK) promoter. YOCK-13 cells produced small amounts of modified insulin and no detectable endogenous L-PK at low glucose concentrations, whereas they produced large amounts of both modified insulin and L-PK in response to high glucose concentrations. Xenotransplantation of YOCK-13 cells via the portal vein into immunosuppressed, totally pancreatectomized pigs decreased hyperglycemia and prolonged survival without adverse effects such as portal thrombosis, liver necrosis, pulmonary embolism, and tumor development. We suggest that this reversibly immortalized, insulin-secreting human hepatocyte line may overcome the shortage of donor pancreata for islet transplantation into patients with type 1 diabetes.

DOI： 10.2337/diabetes.53.1.105

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Propagation of adult rat bone marrow-derived hepatocyte-like cells by serial passages in vitro

M Miyazaki, T Masaka, Akiyama, I, E Nakashima, M Sakaguchi, NH Huh

CELL TRANSPLANTATION   13 ( 4 )   385 - 391   2004

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Previously, we found that hepatocyte growth factor receptor (c-Met)-and alpha-fetoprotein (AFP)-expressing cells were present in adult rat bone marrow, and that these cells also expressed hematopoietic stem cell markers, such as CD34, Thy-1, and c-Kit. When bone marrow cells were cultured in a hepatocyte growth medium (HGM) with HGF and EGF, colonies composed of polygonal cells resembling mature hepatocytes appeared by 2 weeks and grew very slowly because of overgrowth of stromal cells. At days 34-41, 2-mm(2) sheets of hepatocyte-like cells were cut out of their colonies by scratching with an injection needle under observation with a phase contrast microscope, transferred into wells of 24-well plates, and cultured in the HGM medium in the presence or absence of HGF and EGF. When cells reached confluence, cells were detached with trypsin and EDTA and transferred step by step into bigger culture vessels. Thus, hepatocyte-like cells were expanded 1000-fold during less than 4 months. These cells were immunocytochemically stained for albumin and also for AFP and the hematopoietic stem cell markers described above, showing characteristics of oval cells. By RT-PCR, we detected mRNAs of tryptophan-2,3-dioxygenase and tyrosine aminotransferase, markers of hepatocytes at a terminal differentiation stage. The present culture system may be useful for supply of hepatocyte resources for cell transplantation therapy.

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NMR STUDIES OF THE N-TERMINAL SEGMENT OF S100C/A11 PROTEIN

T. Kouno, M. Mizuguchi, M. Sakaguchi, E. Makino, N. H. Huh, K. Kawano

JOURNAL OF PEPTIDE SCIENCE   10   273 - 273   2004

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JOHN WILEY & SONS LTD  

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S100C/A11 is a key mediator of Ca2+-induced growth inhibition of human epidermal keratinocytes

M Sakaguchi, M Miyazaki, M Takaishi, Y Sakaguchi, E Makino, N Kataoka, H Yamada, M Namba, NH Huh

JOURNAL OF CELL BIOLOGY   163 ( 4 )   825 - 835   2003.11

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Language：English   Publisher：ROCKEFELLER UNIV PRESS  

An increase in extracellular Ca2+ induces growth arrest and differentiation of human keratinocytes in culture. We examined possible involvement of S100C/A11 in this growth regulation. On exposure of the cells to high Ca2+, S100C/A11 was specifically phosphorylated at (10)Thr and (94)Ser. Phosphorylation facilitated the binding of S100C/A11 to nucleolin, resulting in nuclear translocation of S100C/A11. In nuclei, S100C/A11 liberated Sp1/3 from nucleolin. The resulting free Sp1/3 transcriptionally activated p21(CIP1/WAF1), a representative negative regulator of cell growth. Introduction of anti-S100C/A11 antibody into the cells largely abolished the growth inhibition induced by Ca2+ and the induction of p21(CIP1/WAF1). In the human epidermis, S100C/A11 was detected in nuclei of differentiating cells in the suprabasal layers, but not in nuclei of proliferating cells in the basal layer. These results indicate that S100C/A11 is a key mediator of the Ca2+-induced growth inhibition of human keratinocytes in culture, and that it may be possibly involved in the growth regulation in vivo as well.

DOI： 10.1083/jcb.200304017

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Dynamic alteration of human beta-defensin 2 localization from cytoplasm to intercellular space in psoriatic skin

WK Huh, T Oono, Y Shirafuji, H Akiyama, J Arata, M Sakaguchi, NH Huh, K Iwatsuki

JOURNAL OF MOLECULAR MEDICINE-JMM   80 ( 10 )   678 - 684   2002.10

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Defensins are cationic antimicrobial peptides with a broad spectrum. Recently human P-defensin 2 (hBD-2) has been isolated from psoriatic skin; however, its exact localization and fate have not been fully under,stood. We studied the distribution pattern of hBD-2 in skin tissues of psoriasis and other inflammatory skin diseases. In the upper spinous and granular layer of psoriasis vulgaris hBD-2 was present in the cytoplasm. In the horny layer the positive signals were in a basket-weave pattern, indicating possible accumulation of hBD-2 in the intercellular space. The similar pattern of hBD-2 distribution was observed in the lesions of nummular eczema and atopic dermatitis. hBD-2 was not detected in the section of normal elbow and knee skin. When isolated psoriatic scales were stained. hBD-2 was detected in a wrapping paper-like distribution pattern surrounding the corneocytes. In horny layer of psoriatic skin hBD-2 was closely associated or c localized with elafin, which is known to be in extracellular space, as demonstrated by double staining. Western blot analysis using cultured human keratinocytes detected hBD-2 with an expected size in the conditioned medium and in the cell lysates when stimulated with 5% FCS or IL-alpha. These results indicate that hBD-2 was synthesized and remained in cytoplasm in the upper spinous and granular layer, and then secreted into intercellular space in the horny layer. This dynamic change in hBD-2 distribution in epidermis is certainly relevant to function as an innate host defense mechanism against invading microorganisms.

DOI： 10.1007/s00109-002-0373-z

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新規タンパク質リン酸化酵素BRPKの解析

阪口 政清, 洪 梅, 片岡 健, 宮崎 正博, 許 南浩

日本癌学会総会記事   61回   81 - 81   2002.10

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Improvement in the differentiated hepatic phenotype of immortalized human hepatocytes by adenovirus mediated p21 gene transfer

N Kobayashi, M Sakaguchi, T Okitsu, T Totsugawa, M Maruyama, T Matsumura, T Watanabe, H Noguchi, Y Kosaka, T Fujiwara, N Tanaka

ASAIO JOURNAL   48 ( 4 )   355 - 359   2002.7

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The p21 molecule, a potent cyclin dependent kinase inhibitor, regulates the transition from the G1 phase to the S phase of the cell cycle and is involved in terminal cellular differentiation. The overexpression of p21 has been shown to induce differentiation in various cell lines. We have made an effort to establish a reliable human hepatocyte cell line as a source of hepatic function in bioartificial liver (BAL) therapy. In this work, we investigated the effect of p21 on the differential phenotype of simian virus 40 large T antigen (SV40Tag) immortalized human hepatocytic NKNT-3 cells. A recombinant adenoviral vector expressing a p21 gene under control of the cytomegalovirus (CMV) promoter (Ad-p21) was used to efficiently transfer genes into NKNT-3 cells. The morphologic alterations, the cell cycle progression, and the expression of p-450 associated enzymes (CYPs) were carefully examined in NKNT-3 cells that had been infected with Ad-p21. Adenovirus mediated gene delivery of p21 was efficiently achieved in NKNT-3 cells without affecting cellular structure. After Ad-p21 infection, NKNT-3 cells were GO/G1 arrested in cell cycle analysis. NKNT-3 cells that had been infected with Ad-p21 showed differentiated hepatic phenotypes in morphology and improvement in protein expression of CYP 3A4 and CYP 2C9. In the present work, we demonstrate that the exogenous expression of p21 enhances the differential phenotype of immortalized hepatocytic NKNT-3 cells.

DOI： 10.1097/00002480-200207000-00005

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Reversible immortalization of human hepatocytes using the Cre/Loxp system for cell therapies

T Totsugawa, N Kobayashi, T Okitsu, H Noguchi, T Watanabe, T Matsumura, M Maruyama, M Hikida, A Ohmori, M Sakaguchi, KA Westerman, P Leboulch, N Tanaka

GASTROENTEROLOGY   123 ( 1 )   54 - 54   2002.7

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Controlled expansion of human endothelial cell Populations by cre-loxP-based reversible immortalization

H Noguchi, N Kobayashi, KA Westerman, M Sakaguchi, T Okitsu, T Totsugawa, T Watanabe, T Matsumura, T Fujiwara, T Ueda, M Miyazaki, N Tanaka, P Leboulch

HUMAN GENE THERAPY   13 ( 2 )   321 - 334   2002.1

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Endothelial cells (ECs) play multiple physiological functions and are central to many pathological processes. Various biological studies as well as cell and gene therapy applications would benefit substantially from a procedure that would result in the expansion in culture of large numbers of highly differentiated human ECs. Here, we report the amplification in vitro of human EC populations, which occurred during the first phase of reversible immortalization resulting from the retroviral transfer of an oncogene that was subsequently excised by Cre-loxP-mediated site-specific recombination. Human umbilical vein endothelial cells (HUVECs) and human liver sinusoidal endothelial cells (HLSECs) were transduced with a retroviral vector that expresses the simian virus 40 large T (SV40T) gene flanked by positive and negative selectable markers and a pair of loxP recombination targets. Transduced HUVECs and HLSECs yielded clones with greatly extended life spans, referred to as HNNT-1 and HNNT-2 cells, respectively. HNNT-1 and HNNT-2 cells showed morphological characteristics of ECs and were maintained in culture up to population doubling level (PDL) 80 for HNNT-1 and PDL 65 for HNNT-2 cells. HNNT-1 and HNNT-2 cells were not tumorigenic when transplanted into severe combined immunodeficiency mice and were sensitive to ganciclovir as well as G418. Both cell clones expressed EC markers, which include factor VIII, VEGF receptors (Flt-1 and KDR/Flk-1), and CD34, and endocytosed acetylated low-density lipoproteins. Formation of capillary-like structures in a Matrigel assay was observed with HNNT-1 and HNNT-2 cells until at least PDL 50. Complete elimination of the transferred SV40T gene was achieved in virtually 100% of HNNT-1 and HNNT-2 cells after infection with a recombinant adenovirus expressing the Cre recombinase fused to a nuclear localization signal and subsequent selection with G418. Reverted cells maintained their differentiated EC phenotype. This study extends the utility of the reversible immortalization procedure and provides a means to expand primary human ECs of various sources for basic studies and possible cell and gene therapies.

DOI： 10.1089/10430340252769833

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レンチウイルスベクターシステム樹立に伴う安全配慮.

都津川敏範, 小林直哉, 丸山昌伸, 小坂芳和, 興津 輝, 荒田 尚, 阪口政清, 藤原俊義, 倉林 譲, 田中紀章

Organ Biology   9,4,357-364   2002

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Lentiviral transfer of the LacZ gene into human endothelial cells and human bone marrow mesenchymal stem cells

T Totsugawa, N Kobayashi, T Okitsu, H Noguchi, T Watanabe, T Matsumura, M Maruyama, T Fujiwara, M Sakaguchi, N Tanaka

CELL TRANSPLANTATION   11 ( 5 )   481 - 488   2002

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Because one of the attractive characteristics of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors is that it can infect even nondividing cells, a lentivirus-mediated gene delivery system is currently being paid a great deal of attention as an innovative tool for gene transfer into target cells. The purpose of the work was to investigate the efficacy of lentiviral transfer of the LacZ gene into human umbilical vein endothelial cells (HUVECs) and human bone marrow mesenchymal stem cells (HMSCs) in vitro. For the present study, a vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector encoding the E. coli LacZ gene tagged with nuclear localization signal (NLS) was generated in 293T cells by means of the three-plasmid system. The resulting lentiviral vector, LtV-NLS/LacZ, was allowed to infect HUVECs and HMSCs. Approximately 70% of HUVECs were positive for LacZ expression and 50% of HMSCs showed LacZ activity. There was no significant difference in transduction efficacy between early and late-passage phases in both cells. LtV-NLS/LacZ-transduced HUVECs showed gene expression of endothelial markers including CD34 and flt-1 and KDR/flk-1 of vascular endothelial growth factor (VEGF) receptors and had angiogenic potential as efficiently as primarily cultured HUVECs in a Matrigel assay. These findings provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in human endothelial cells and stem cells that could be useful for tissue engineering.

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Transduction of immortalized human hepatocytes with p21 to enhance differentiated phenotypes

T Kunieda, N Kobayashi, M Sakaguchi, T Okitsu, T Totsugawa, T Watanabe, T Matsumura, M Maruyama, H Noguchi, M Takesue, N Shibata, K Ohmoto, T Fujiwara, S Yamamoto, N Tanaka

CELL TRANSPLANTATION   11 ( 5 )   421 - 428   2002

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We previously constructed an immortal human hepatocyte line NKNT-3 with a simian virus 40 T antigen (SV40T) to develop cell-based biological therapies. p21 is a molecule that regulates the transition from the G, phase to the S phase of the cell cycle. Investigators have demonstrated that overexpression of p21 induces differentiation in various cell lines. In the current study we examined the effect of p21 on differentiated phenotypes of SV40T-immortalized NKNT-3 cells. A replication-deficient adenovirus vector expressing a human wild-type p21 cDNA under the control of the CMV promoter (Ad5CMVp21) and a human wild-type p21 protein fused to the protein transduction domain from the human immunodeficiency virus (HIV) TAT protein (TAT/p21) were utilized to achieve efficient delivery of p21 into NKNT-3 cells. Morphological alterations, cell cycle progression, and expression of albumin and p-450 associated enzymes (CYPs) 3A4 and 2C9 were evaluated in NKNT-3 cells treated with Ad5CMVp21 and TAT/p21. Efficient adenovirus-based p21 transfer and TAT-mediated p21 protein delivery were confirmed in NKNT-3 cells in an immunofluorescence study and Western blotting analysis. Transduction of NKNT-3 cells with p21 predominantly arrested the cell cycle at the G, checkpoint, resulting in differentiated hepatic phenotypes in morphology and improvement in protein expression of albumin, CYP 3A4, and CYP C29. We here show that exogenous expression of p21 augments cellular differentiation in immortalized human NKNT-3 cells.

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Reactivation of liver-specific gene expression in an immortalized human hepatocyte cell line by introduction of the human HNF4 alpha 2 gene

Y Inoue, M Miyazaki, T Tsuji, M Sakaguchi, K Fukaya, NH Huh, M Namba

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE   8 ( 5 )   481 - 487   2001.11

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Language：English   Publisher：PROFESSOR D A SPANDIDOS  

An immortalized human hepatocyte cell line (OUMS-29) was established from fetal liver by transfection with the SV-40 large T antigen gene that has certain liver-specific functions such as albumin production and enzyme activities of CYP1A1, 1A2, and 2E1. To make OUMS-29 cells express other liver-specific functions, the human hepatocyte nuclear factor 4 alpha2 (HNF4 alpha2) gene was introduced into the cells, because this gene was found to be markedly down-regulated. The transduced HNF4 alpha2 was overexpressed in the nuclei of the transfected cells, and its DNA-binding activity was also detected. The liver-specific genes such as apolipoprotein AI, CII, CIII, blood coagulation factor X, alpha1-antitrypsin, and HNF1 alpha were up-regulated. Thus, this cell line is expected to be a useful tool for studying the differentiated human hepatocyte, functions.

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Adenovirus-mediated p21 gene transfer enhances differentiated hepatic phenotypes of immortalized human hepatocytes.

N Kobayashi, H Noguchi, T Totsugawa, T Watanabe, T Matsumura, M Maruyama, T Matsumoto, T Fujimara, N Tanaka, M Sakaguchi

HEPATOLOGY   34 ( 4 )   305A - 305A   2001.10

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：W B SAUNDERS CO  

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Expansion of human hepatocyte populations by a retroviral gene transfer of simian virus 40 large T antigen

N Kobayashi, KA Westerman, T Taguchi, M Sakaguchi, T Fujiwara, H Urata, N Kishimoto, N Hayashi, S Nakaji, T Murakami, P Leboulch, N Tanaka

ASAIO JOURNAL   47 ( 5 )   481 - 485   2001.9

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Language：English   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

A hybrid artificial liver (HAL) could be used to treat acute liver failure or to serve as a temporary support until orthotopic liver transplantation is available. Primary human hepatocytes are ideal as a source of hepatic function in a HAL device. However, the worldwide shortage of human livers available for hepatocyte isolation severely limits this form of therapy. A possible alternative is to use a tightly regulated cell line that can be economically grown in culture to have differentiated liver function. In this work, human hepatocytes were immortalized with a retroviral vector SSR#69 expressing the genes of simian virus 40 large T antigen and herpes simplex virus-thymidine kinase. One of the resulting clones, NKNT-3, showed the gene expression of differentiated liver function and were sensitive to the antiviral agent ganciclovir. When transplanted into the spleen of rats subjected to 90% hepatectomy, NKNT-3 cells prolonged the survival of 90% hepatectomized rats. The cells provide the advantages of unlimited availability, sterility, uniformity, and freedom from pathogens. This work represents a potential novel strategy for resolving the organ shortage that currently limits the use of primary human hepatocytes to develop a HAL.

DOI： 10.1097/00002480-200109000-00017

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Construction of a differentiated human hepatocyte cell line expressing the herpes simplex virus-thymidine kinase gene

N Kobayashi, M Miyazaki, KA Westerman, H Noguchi, M Sakaguchi, T Totsugawa, T Watanabe, T Matsumura, T Fujiwara, P Leboulch, N Tanaka, M Namba

ASAIO JOURNAL   47 ( 5 )   476 - 480   2001.9

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Transient support using a hybrid artificial liver (HAL) device is a promising treatment for the patients with acute liver failure. Primary human hepatocytes are an ideal source for HAL therapy; however, the number of human livers available for hepatocyte isolation is limited by competition for use in whole organ transplantation. To overcome this problem, we previously established a highly differentiated human fetal hepatocyte cell line OUMS-29. Considering the potential risk when using these genetically engineered cells in humans, additional safeguards should be added to make the cells more clinically useful. In this work, the herpes simplex virus thymidine kinase (HSVtk) gene was retrovirally introduced into OUMS-29 cells. One of the HSVtk-expressed clones, OUMS-29/thymidine kinase (TK), grew in chemically defined serum free medium and expressed the genes of albumin, asialoglycoprotein receptor, glutamine synthetase, glutathione-S-transferase pi, and blood coagulation factor X. In vitro sensitivity of the cells to ganciclovir was evaluated. Intrasplenic transplantation of 50 X 10(6) OUMS-29/TK cells prolonged the survival of 90% hepatectomized rats compared with medium injection alone (control). In the present study, we have established highly differentiated immortalized human hepatocytes with tight regulation. The cells may be clinically useful for HAL treatment.

DOI： 10.1097/00002480-200109000-00016

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S100Cタンパク質のアクチンとの結合及びその意義

阪口 政清, 宮崎 正博, 難波 正義, 許 南浩

Tissue culture research communications : the journal of experimental & applied cell culture research = 組織培養研究   20 ( 2 )   108 - 108   2001.7

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レンチウイルスベクターによるヒト血管内皮細胞への効果的な遺伝子導入.

都津川敏範, 小林直哉, 丸山昌伸, 興津 輝, 野口洋文, 松村年久, 渡辺剛正, 阪口政清, 藤原俊義, 田中紀章

Organ Biology   2001

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ヒト線維芽細胞の不死化, 癌化にともなう細胞内タンパク質の変化 : 酸化還元系酵素の発現の変化について

近藤 格, 阪口 政清, 難波 正義

生物物理化学 = Journal of Electrophoresis   44   35 - 35   2000.10

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Effects of Oxygen Concentrations on Human Fibroblasts Treated with Fe^<3+>-NTA

PU Hong, SAKAGUCHI Masakiyo, KONDO Tadashi, KONDO Asami, JIANG H-X., KAWABATA Teruyuki, NAMBA Masayoshi

19 ( 2 )   78 - 78   2000.6

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Relationship between Contact Inhibition and Intranuclear S100C of Normal Human Fibroblasts

SAKAGUCHI Masakiyo, MIYAZAKI Masahiro, KOUCHI Hirosuke, KONDO Tadashi, NAMBA Masayoshi

19 ( 2 )   87 - 87   2000.6

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【臓器移植と医工学 その現況と展望】 急性肝不全に対する不死化ヒト肝細胞の移植

小林 直哉, 野口 洋文, 田中 紀章, 深谷 憲一, 阪口 政清, 井上 祐介, 宮崎 正博, 難波 正義

今日の移植   13 ( 3 )   216 - 221   2000.5

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SF10-3 不死化ヒト臍帯静脈内皮細胞株(HNKT-1)の樹立

野口 洋文, 小林 直哉, 宮崎 正博, 井上 祐介, 阪口 政清, 藤原 俊義, 田中 紀章

日本外科学会雑誌   101   129 - 129   2000.3

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Language：Japanese   Publisher：一般社団法人日本外科学会  

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ペプシンを産生するラット胃粘膜上皮細胞株(OUMS-37)の樹立と分化誘導の検討

濮 紅, 湯浅 貴恵, 近藤 麻美, 阪口 政清, 高 崇, 稲田 憲一, 難波 正義

Tissue culture research communications : the journal of experimental & applied cell culture research = 組織培養研究   18 ( 1 )   63 - 63   1999.3

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Language：Japanese  

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細胞外ATPは不死化ヒト線維芽細胞の増殖をヒト正常線維芽細胞に比べより強く抑制する

井上 裕介, 阪口 政清, 難波 正義

Tissue culture research communications : the journal of experimental & applied cell culture research = 組織培養研究   18 ( 1 )   67 - 67   1999.3

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P-986 ラット急性肝不全モデルにおける不死化胎児肝細胞OUMS-29の脾臓内移植の効果

小林 直哉, 宮崎 正博, 井上 裕介, 阪口 政清, 近藤 麻美, 田中 紀章, 難波 正義

日本外科学会雑誌   100   558 - 558   1999.2

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Language：Japanese   Publisher：一般社団法人日本外科学会  

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ATP inhibition of proliferation of immortalized human fibroblasts is greater than that of normal human diploid fibroblasts

B Katayama, M Sakaguchi, JW Li, H Pu, Y Inoue, M Namba

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE   2 ( 5 )   603 - 606   1998.11

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Language：English   Publisher：PROFESSOR D A SPANDIDOS  

It is known that cancers develop by a multi-step process. Normal cells are first immortalized, and then transformed into tumorigenic cells. Normal human cells are very rarely immortalized, but once they are, they are relatively easily transformed into tumorigenic cells. This indicates that the immortalization step plays a critical part in the development of human cancers. Thus, elucidation of the mechanisms of this step would shed light on the process of carcinogenesis in human cells. To understand the causes of immortalization, it is important to determine the differences in cellular phenotype between immortalized and normal human cells. In this study, we found that immortalized human fibroblasts were more sensitive to the growth inhibitory effects of ATP than normal human fibroblasts. ADP was as effective as ATP, but AMP, adenosine, and phosphoric acid were not. These results indicate that a high-energy bound of ATP and ADP may contribute to the growth inhibition of the cells. When the immortalized cells were pulse-labeled with [P-32]-ATP, 30-, 31-, 33- and 40-kDa membrane fraction proteins were more prominently labeled in the immortalized cells than in the normal cells. At present, the characteristics of these proteins are being investigated.

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Differential intracellular localization of transferrin produced in human fibroblasts and that incorporated from culture medium

SAKAGUCHI M., KONDO T., NAMBA M.

17 ( 1 )   46 - 46   1998.3

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