研究者詳細 - 阪口　政清

論文 - 阪口　政清

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Antithrombin regulates neutrophil activities through the stimulation of C-type lectin family 1A 査読

Yohei Takahashi, Soe Soe Htwe, Dengli Wang, Hidenori Wake, Mariko Yata, Nahoko Tomonobu, Rie Kinoshita, Masakiyo Sakaguchi, Masahiro Nishibori

Blood Vessels, Thrombosis & Hemostasis 2025年2月

An S100A8/A9 Neutralizing Antibody Potently Ameliorates Contact Hypersensitivity and Atopic Dermatitis Symptoms. 国際誌

Yuma Gohara, Rie Kinoshita, Nahoko Tomonobu, Fan Jiang, Yukiko Matsunaga, Yuki Hashimoto, Tomoko Honjo, Ken-Ichi Yamamoto, Hitoshi Murata, Toshiki Ochi, Ni Luh Gede Yoni Komalasari, Akira Yamauchi, Futoshi Kuribayashi, Yoshihiko Sakaguchi, Junichiro Futami, Yusuke Inoue, Eisaku Kondo, Shinichi Toyooka, Shin Morizane, Akira Ishiko, Shigeru Morita, Kazumi Sagayama, Kenichiro Nakao, Masakiyo Sakaguchi

The Journal of investigative dermatology 2025年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Contact hypersensitivity and atopic dermatitis are pervasive inflammatory skin diseases with similar symptoms, and their global prevalence is steadily increasing. Many compounds and biotics have been developed to target molecules critical to the etiology or pathogenesis of contact hypersensitivity and atopic dermatitis. However, these molecules are sometimes ineffective or lose their potency during the therapeutic course. Therefore, innovative medicines are still needed for the treatment of intractable cases. We focused on S100A8/A9, a heterodimer complex of S100A8 and S100A9 that is abundant in the extracellular milieu of inflammatory skin lesions. Although S100A8/A9 is primarily recognized as a diagnostic marker protein, we have previously shown that it also plays a crucial role in contact hypersensitivity and atopic dermatitis progression. This insight inspired us to develop its inhibitory antibody, leading to the ground-breaking Ab45. In this study, we demonstrated that Ab45 effectively prevented disease symptoms in various models and that its disease-ameliorating activity likely involved the downregulation of several disease-relevant molecules, including Il-23a, Il-36g, S100a8, and S100a9. We also created a humanized version of Ab45, HuAb45, which exhibited similar effectiveness. These antibodies show great promise for the treatment of contact hypersensitivity and atopic dermatitis and possibly for other inflammatory skin diseases.

DOI： 10.1016/j.jid.2025.01.007

Expression of SPRED2 in the lung adenocarcinoma. 査読国際誌

Yoko Ota, Tong Gao, Masayoshi Fujisawa, I Wayan Sumardika, Masakiyo Sakaguchi, Shinichi Toyooka, Teizo Yoshimura, Akihiro Matsukawa

Pathology, research and practice 265 155721 - 155721 2025年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

SPRED2 (Sprouty-related, EVH1 domain-containing protein 2), a negative regulator of the ERK1/2 pathway, is downregulated in several cancers; however, the significance of SPRED2 expression in lung adenocarcinoma (LUAD) remains unclear. Here, we investigated the pathological expression of SPRED2 and its relationship with ERK1/2 activation (ERK1/2 phosphorylation), Ki67 index and clinicopathological features in 77 LUAD tissues from clinical patients. Immunohistochemically, SPRED2 expression was decreased in invasive adenocarcinoma (IA) compared to adenocarcinoma in situ (AIS). There was a negative correlation between SPRED2 expression and pERK1/2 levels and a positive correlation between SPRED2 expression and Ki67 index. In the database analysis, the survival probability was higher in patients with higher SPRED2 expression than in those with lower expression. In vitro, SPRED2 deletion increased cell proliferation, migration and invasion of three LUAD cell lines (A549:KRAS mutation, H1993:METamplification, and HCC4006:EGFR mutation), whereas SPRED2 overexpression decreased these responses. Thus, SPRED2 appears to be a regulator of LUAD progression and a potential target for the treatment of LUAD.

DOI： 10.1016/j.prp.2024.155721

Exosomal delivery of miR-200b-3p suppresses the growth of hepatocellular carcinoma cells by targeting ERG- and VEGF-mediated angiogenesis. 査読国際誌

Yuze Wang, Aye Moh-Moh-Aung, Tianyi Wang, Masayoshi Fujisawa, Toshiaki Ohara, Ken-Ichi Yamamoto, Masakiyo Sakaguchi, Teizo Yoshimura, Akihiro Matsukawa

Gene 931 148874 - 148874 2024年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

Hepatocellular carcinoma (HCC) remains a lethal malignancy with limited treatment options. Recent discoveries have highlighted the pivotal role of miRNAs in HCC progression. We previously reported that the expression of miR-200b-3p was decreased in HCC cells and exosomal miR-200b-3p from hepatocytes inhibited angiogenesis by suppressing the expression of the endothelial transcription factor ERG (erythroblast transformation-specific (ETS)-related gene), leading to the hypothesis that the delivery of this miRNA may inhibit angiogenesis and suppress HCC growth in vivo. Here, we tested this hypothesis by using human HCC inoculation models. First, we transfected the human HepG2 HCC cells and established a stable cell line that overexpressed a high level of miR-200b-3p. When miR-200b-3p-overexpressing cells were injected into severe combined immunedeficiency (SCID)-beige mice, tumor growth was significantly reduced compared to tumors of control cells, with a reduction in the expression of ERG and vascular endothelial growth factor (VEGF) and subsequent angiogenesis. Intra-tumoral injection of exosomes containing high levels of miR-200b-3p also reduced the growth of parental HepG2 tumors with reduced ERG and VEGF expression and angiogenesis. These results validate the inhibitory role of miR-200b-3p in tumor angiogenesis, thereby suppressing HCC tumor growth, and provide a novel insight into its potential therapeutic application.

DOI： 10.1016/j.gene.2024.148874

NCF-1 plays a pivotal role in the survival of adenocarcinoma cells of pancreatic and gastric origins. 国際誌

Chiemi Furuya-Ikude, Akane Kitta, Naoko Tomonobu, Yoshihiro Kawasaki, Masakiyo Sakaguchi, Eisaku Kondo

In vitro cellular & developmental biology. Animal 60 ( 10 ) 1151 - 1159 2024年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

Reactive oxygen species (ROS) play a pivotal biological role in cells, with ROS function differing depending on cellular conditions and the extracellular environment. Notably, ROS act as cytotoxic factors to eliminate infectious pathogens or promote cell death under cellular stress, while also facilitating cell growth (via ROS-sensing pathways) by modifying gene expression. Among ROS-related genes, neutrophil cytosolic factor-1 (NCF-1; p47phox) was identified as a ROS generator in neutrophils. This product is a subunit of a cytosolic NADPH oxidase complex activated in response to pathogens such as bacteria and viruses. NCF-1 has been examined primarily in terms of ROS-production pathways in macrophages and neutrophils; however, the expression of this protein and its biological role in cancer cells remain unclear. Here, we report expression of NCF-1 in pancreatic and gastric cancers, and demonstrate its biological significance in these tumor cells. Abundant expression of NCF-1 was observed in pancreatic adenocarcinoma (PDAC) lines and in patient tissues, as well as in gastric adenocarcinomas. Accumulation of the protein was also detected in the invasive/metastatic foci of these tumors. Unexpectedly, BxPC-3 underwent apoptotic cell death when transfected with a small interfering RNA (siRNA) specific to NCF-1, whereas the cells treated with a control siRNA proliferated in a time-dependent manner. A similar phenomenon was observed in HSC-58, a poorly differentiated gastric adenocarcinoma line. Consequently, the tumor cells highly expressing NCF-1 obtained coincident accumulation of ROS and reduced glutathione (GSH) with expression of glutathione peroxidase 4 (GPX4), a quencher involved in ferroptosis. Unlike the conventional role of ROS as a representative cytotoxic factor, these findings suggest that NCF-1-mediated ROS generation may be required for expansive growth of PDAC and gastric cancers.

DOI： 10.1007/s11626-024-00994-0

Enhanced design of pCMViR-TSC plasmid vector for sustainably high cargo gene expression in mammalian cells. 査読国際共著国際誌

Masakiyo Sakaguchi, Rie Kinoshita, Nahoko Tomonobu, Yoshihiko Sakaguchi, Junichiro Futami, Akira Yamauchi, Hitoshi Murata, Ken-Ichi Yamamoto, Tetta Takahashi, Yuma Gohara, Toshiki Ochi, Fan Jiang, Ni Luh Gede Yoni Komalasari, Youyi Chen, I Made Winarsa Ruma, I Wayan Sumardika, Jin Zhou, Tomoko Honjo, Futoshi Kuribayashi, Kazumi Sagayama, Shinichi Toyooka, Eisaku Kondo, Yusuke Inoue

In vitro cellular & developmental biology. Animal 2024年11月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）

The first-generation pCMViR-TSC, implemented through the promoter sandwich rule, yields 10- to 100-fold higher gene expression than the standard plasmid used with the CMV (cytomegalovirus) or CAG promoter. However, the vector's shortcomings limit its utility to transient expression only, as it is not suitable for establishing stable transformants in mammalian cells. To overcome this weakness, we here introduce the improved plasmid vector pSAKA-4B, derived from pCMViR-TSC as a second-generation chromosome-insertable vector. This vector facilitates the linear entry of the expression unit into the TTAA site of DNA universally with transposase assistance. The vector is helpful for the indefinite expression of our target gene. The new vector system is proven here to be efficient in establishing stable transformants with a high likelihood of positive clones that exhibit significantly elevated expression levels of the delivered foreign gene. This system, alongside the first-generation vector, is therefore instrumental for diverse basic research endeavors concerning genes, proteins, cells, and animals, and potentially for clinical applications such as gene therapy.

DOI： 10.1007/s11626-024-00992-2

Pathological and Biological Significance of the Specific Glycan, TRA-1-60, on Aggressive Gastric Adenocarcinoma. 査読国際誌

Ayaka Mitsui, Hidekazu Iioka, Yiwei Ling, Shujiro Okuda, Akira Kurose, Michael Schopperle, Tomoko Kondo, Masakiyo Sakaguchi, Ken Saito, Eisaku Kondo

Laboratory investigation; a journal of technical methods and pathology 104 ( 7 ) 102073 - 102073 2024年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier BV

The glycans form a unique complex on the surface of cancer cells and play a pivotal role in tumor progression, impacting proliferation, invasion, and metastasis. TRA-1-60 is a glycan that was identified as a critical marker for the establishment of fully reprogrammed inducible pluripotent stem cells. Its expression has been detected in multiple cancer tissues, including embryonal carcinoma, prostate cancer, and pancreatic cancer, but the biological and pathological characterization of TRA-1-60-expressing tumor cells remains unclear within various types of malignancies. Here, we report the biological characteristics of TRA-1-60-expressing gastric cancer cells, especially those with its cell surface expression, and the therapeutic significance of targeting TRA-1-60. The cells with cell membrane expression of TRA-1-60 were mainly observed in the invasive area of patient gastric cancer tissues and correlated with advanced stages of the disease based on histopathological and clinicopathological analyses. In vitro analysis using a scirrhous gastric adenocarcinoma line, HSC-58, which highly expresses TRA-1-60 on its plasma membrane, revealed increased stress-resistant mechanisms, supported by the upregulation of glutathione synthetase and NCF-1 (p47phox) via lipid-ROS regulatory pathways, as detected by RNA-seq analysis followed by oxidative stress gene profiling. Our in vivo therapeutic study using the TRA-1-60-targeting antibody-drug conjugate, namely, Bstrongomab-conjugated monomethyl auristatin E, showed robust efficacy in a mouse model of peritoneal carcinomatosis induced by intraperitoneal xenograft of HSC-58, by markedly reducing massive tumor ascites. Thus, targeting the specific cell surface glycan, TRA-1-60, shows a significant therapeutic impact in advanced-stage gastric cancers.

DOI： 10.1016/j.labinv.2024.102073

Correction: S100A11 is involved in the progression of colorectal cancer through the desmosome-catenin-TCF signaling pathway. 査読国際共著国際誌

Jin Zhou, Hitoshi Murata, Nahoko Tomonobu, Naoko Mizuta, Atsuko Yamakawa, Ken-Ichi Yamamoto, Rie Kinoshita, Masakiyo Sakaguchi

In vitro cellular & developmental biology. Animal 2024年6月

SPRED2 Is a Novel Regulator of Autophagy in Hepatocellular Carcinoma Cells and Normal Hepatocytes. 査読国際誌

Tianyi Wang, Tong Gao, Masayoshi Fujisawa, Toshiaki Ohara, Masakiyo Sakaguchi, Teizo Yoshimura, Akihiro Matsukawa

International journal of molecular sciences 25 ( 11 ) 2024年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

Sprouty-related enabled/vasodilator-stimulated phosphoprotein homology 1 domain containing 2 (SPRED2) is an inhibitor of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway and has been shown to promote autophagy in several cancers. Here, we aimed to determine whether SPRED2 plays a role in autophagy in hepatocellular carcinoma (HCC) cells. The Cancer Genome Atlas (TCGA) Liver Cancer Database showed a negative association between the level of SPRED2 and p62, a ubiquitin-binding scaffold protein that accumulates when autophagy is inhibited. Immunohistochemically, accumulation of p62 was detected in human HCC tissues with low SPRED2 expression. Overexpression of SPRED2 in HCC cells increased the number of autophagosomes and autophagic vacuoles containing damaged mitochondria, decreased p62 levels, and increased levels of light-chain-3 (LC3)-II, an autophagy marker. In contrast, SPRED2 deficiency increased p62 levels and decreased LC3-II levels. SPRED2 expression levels were negatively correlated with translocase of outer mitochondrial membrane 20 (TOM20) expression levels, suggesting its role in mitophagy. Mechanistically, SPRED2 overexpression reduced ERK activation followed by the mechanistic or mammalian target of rapamycin complex 1 (mTORC1)-mediated signaling pathway, and SPRED2 deficiency showed the opposite pattern. Finally, hepatic autophagy was impaired in the liver of SPRED2-deficient mice with hepatic lipid droplet accumulation in response to starvation. These results indicate that SPRED2 is a critical regulator of autophagy not only in HCC cells, but also in hepatocytes, and thus the manipulation of this process may provide new insights into liver pathology.

DOI： 10.3390/ijms25116269

S100A11 is involved in the progression of colorectal cancer through the desmosome-catenin-TCF signaling pathway. 査読国際誌

Jin Zhou, Hitoshi Murata, Nahoko Tomonobu, Naoko Mizuta, Atsuko Yamakawa, Ken-Ichi Yamamoto, Rie Kinoshita, Masakiyo Sakaguchi

In vitro cellular & developmental biology. Animal 2024年6月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Compiling evidence has indicated that S100A11 expression at high levels is closely associated with various cancer species. Consistent with the results reported elsewhere, we have also revealed that S100A11 is highly expressed in squamous cell carcinoma, mesothelioma, and pancreatic cancers and plays a crucial role in cancer progression when secreted into extracellular fluid. Those studies are all focused on the extracellular role of S100A11. However, most of S100A11 is still present within cancer cells, although the intracellular role of S100A11 in cancer cells has not been fully elucidated. Thus, we aimed to investigate S100A11 functions within cancer cells, primarily focusing on colorectal cancer cells, whose S100A11 is abundantly present in cells and still poorly studied cancer for the protein. Our efforts revealed that overexpression of S100A11 promotes proliferation and migration, and downregulation inversely dampens those cancer behaviors. To clarify how intracellular S100A11 aids cancer cell activation, we tried to identify S100A11 binding proteins, resulting in novel binding partners in the inner membrane, many of which are desmosome proteins. Our molecular approach defined that S100A11 regulates the expression level of DSG1, a component protein of desmosome, by which S100A11 activates the TCF pathway via promoting nuclear translocation of γ-catenin from the desmosome. The identified new pathway greatly helps to comprehend S100A11's nature in colorectal cancers and others.

DOI： 10.1007/s11626-024-00930-2

PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve sheath tumours. 査読国際誌

Shota Takihira, Daisuke Yamada, Tatsunori Osone, Tomoka Takao, Masakiyo Sakaguchi, Michiyuki Hakozaki, Takuto Itano, Eiji Nakata, Tomohiro Fujiwara, Toshiyuki Kunisada, Toshifumi Ozaki, Takeshi Takarada

British journal of cancer 130 ( 9 ) 1493 - 1504 2024年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: Paired related-homeobox 1 (PRRX1) is a transcription factor in the regulation of developmental morphogenetic processes. There is growing evidence that PRRX1 is highly expressed in certain cancers and is critically involved in human survival prognosis. However, the molecular mechanism of PRRX1 in cancer malignancy remains to be elucidated. METHODS: PRRX1 expression in human Malignant peripheral nerve sheath tumours (MPNSTs) samples was detected immunohistochemically to evaluate survival prognosis. MPNST models with PRRX1 gene knockdown or overexpression were constructed in vitro and the phenotype of MPNST cells was evaluated. Bioinformatics analysis combined with co-immunoprecipitation, mass spectrometry, RNA-seq and structural prediction were used to identify proteins interacting with PRRX1. RESULTS: High expression of PRRX1 was associated with a poor prognosis for MPNST. PRRX1 knockdown suppressed the tumorigenic potential. PRRX1 overexpressed in MPNSTs directly interacts with topoisomerase 2 A (TOP2A) to cooperatively promote epithelial-mesenchymal transition and increase expression of tumour malignancy-related gene sets including mTORC1, KRAS and SRC signalling pathways. Etoposide, a TOP2A inhibitor used in the treatment of MPNST, may exhibit one of its anticancer effects by inhibiting the PRRX1-TOP2A interaction. CONCLUSION: Targeting the PRRX1-TOP2A interaction in malignant tumours with high PRRX1 expression might provide a novel tumour-selective therapeutic strategy.

DOI： 10.1038/s41416-024-02632-8

Dissection of the signal transduction machinery responsible for the lysyl oxidase-like 4-mediated increase in invasive motility in triple-negative breast cancer cells: mechanistic insight into the integrin-β1-NF-κB-MMP9 axis. 査読国際共著国際誌

Fan Jiang, Youyi Chen, Nahoko Tomonobu, Rie Kinoshita, Ni Luh Gede Yoni Komalasari, Carlos Ichiro Kasano-Camones, Kazumi Ninomiya, Hitoshi Murata, Ken-Ichi Yamamoto, Yuma Gohara, Toshiki Ochi, I Made Winarsa Ruma, I Wayan Sumardika, Jin Zhou, Tomoko Honjo, Yoshihiko Sakaguchi, Akira Yamauchi, Futoshi Kuribayashi, Junichiro Futami, Eisaku Kondo, Yusuke Inoue, Shinichi Toyooka, Masakiyo Sakaguchi

Frontiers in oncology 14 ( 1371307 ) 1371307 - 1371307 2024年5月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: Triple-negative breast cancer (TNBC) cells are a highly formidable cancer to treat. Nonetheless, by continued investigation into the molecular biology underlying the complex regulation of TNBC cell activity, vulnerabilities can be exposed as potential therapeutic targets at the molecular level. We previously revealed that lysyl oxidase-like 4 (LOXL4) promotes the invasiveness of TNBC cells via cell surface annexin A2 as a novel binding substrate of LOXL4, which promotes the abundant localization of integrin-β1 at the cancer plasma membrane. However, it has yet to be uncovered how the LOXL4-mediated abundance of integrin-β1 hastens the invasive outgrowth of TNBC cells at the molecular level. METHODS: LOXL4-overexpressing stable clones were established from MDA-MB-231 cells and subjected to molecular analyses, real-time qPCR and zymography to clarify their invasiveness, signal transduction, and matrix metalloprotease (MMP) activity, respectively. RESULTS: Our results show that LOXL4 potently promotes the induction of matrix metalloprotease 9 (MMP9) via activation of nuclear factor-κB (NF-κB). Our molecular analysis revealed that TNF receptor-associated factor 4 (TRAF4) and TGF-β activated kinase 1 (TAK1) were required for the activation of NF-κB through Iκβ kinase kinase (IKKα/β) phosphorylation. CONCLUSION: Our results demonstrate that the newly identified LOXL4-mediated axis, integrin-β1-TRAF4-TAK1-IKKα/β-Iκβα-NF-κB-MMP9, is crucial for TNBC cell invasiveness.

DOI： 10.3389/fonc.2024.1371307

Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface. 査読国際共著国際誌

Tetta Takahashi, Nahoko Tomonobu, Rie Kinoshita, Ken-Ichi Yamamoto, Hitoshi Murata, Ni Luh Gede Yoni Komalasari, Youyi Chen, Fan Jiang, Yuma Gohara, Toshiki Ochi, I Made Winarsa Ruma, I Wayan Sumardika, Jin Zhou, Tomoko Honjo, Yoshihiko Sakaguchi, Akira Yamauchi, Futoshi Kuribayashi, Eisaku Kondo, Yusuke Inoue, Junichiro Futami, Shinichi Toyooka, Yoshito Zamami, Masakiyo Sakaguchi

Frontiers in oncology 14 1371342 - 1371342 2024年3月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers Media SA

Background

Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-β1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain.

Methods

Cell invasion was assessed using a transwell-based assay, protein–protein interactions by an immunoprecipitation–Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography.

Results

We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion.

Conclusion

We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion.

DOI： 10.3389/fonc.2024.1371342

[The plasma protein HRG is an important factor for preventing sepsis and maintaining homeostatic response]. 査読

Masahiro Nishibori, Hidenori Wake, Masakiyo Sakaguchi

Nihon yakurigaku zasshi. Folia pharmacologica Japonica 159 ( 2 ) 107 - 111 2024年3月

担当区分：責任著者記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Pharmacological Society

Acute phase proteins such as CRP, amyloid protein A, and α1-antitrypsin are produced in the liver and their plasma levels are increased during the acute inflammatory response. In contrast, there are plasma proteins whose dynamics are opposite to acute phase proteins. This group includes histidine-rich glycoprotein (HRG), inter-α-inhibitor proteins, albumin, and transthyretin. HRG binds to a variety of factors and regulates the fundamental processes; the blood coagulation, the clearance of apoptotic cells, and tumor growth. In the present review, we focus on the anti-septic effects of HRG in mice model, the actions of HRG on human blood cells/vascular endothelial cells, and the identification of a novel receptor CLEC1A for HRG, based on our recent findings. HRG appears to maintain the quiescence of neutrophils; a round shape, the low levels of spontaneous release of ROS, the ease passage through artificial microcapillaries, and prevention of adhesion to vascular endothelial cells. HRG also inhibited activation of vascular endothelial cells; the suppression of adhesion molecules and the inhibition of HMGB1 mobilization and cytokine secretion. It was shown that plasma HRG level was an excellent biomarker of septic patients in ICU for the evaluation of severity and prognosis. So far little attention has been paid to HRG in terms of a functional role in sepsis and ARDS, however, it is strongly suggested that HRG may be an important plasma factor that prevents a progress in the septic cascade and maintains the homeostasis of blood cells and vascular endothelial cells.

DOI： 10.1254/fpj.23027

Correction to: Periostin secreted by cancer‑associated fibroblasts promotes cancer progression and drug resistance in non‑small cell lung cancer. 査読国際誌

Fumiaki Takatsu, Ken Suzawa, Shuta Tomida, Yin Min Thu, Masakiyo Sakaguchi, Tomohiro Toji, Masayoshi Ohki, Shimpei Tsudaka, Keiichi Date, Naoki Matsuda, Kazuma Iwata, Yidan Zhu, Kentaro Nakata, Kazuhiko Shien, Hiromasa Yamamoto, Akiko Nakayama, Mikio Okazaki, Seiichiro Sugimoto, Shinichi Toyooka

Journal of molecular medicine (Berlin, Germany) 102 ( 1 ) 147 - 147 2024年1月

Periostin secreted by cancer-associated fibroblasts promotes cancer progression and drug resistance in non-small cell lung cancer. 査読国際誌

Fumiaki Takatsu, Ken Suzawa, Shuta Tomida, Yin Min Thu, Masakiyo Sakaguchi, Tomohiro Toji, Masayoshi Ohki, Shimpei Tsudaka, Keiichi Date, Naoki Matsuda, Kazuma Iwata, Yidan Zhu, Kentaro Nakata, Kazuhiko Shien, Hiromasa Yamamoto, Akiko Nakayama, Mikio Okazaki, Seiichiro Sugimoto, Shinichi Toyooka

Journal of molecular medicine (Berlin, Germany) 101 ( 12 ) 1603 - 1614 2023年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

Cancer-associated fibroblasts (CAFs) are important components in the tumor microenvironment, and we sought to identify effective therapeutic targets in CAFs for non-small cell lung cancer (NSCLC). In this study, we established fibroblast cell lines from the cancerous and non-cancerous parts of surgical lung specimens from patients with NSCLC and evaluated the differences in behaviors towards NSCLC cells. RNA sequencing analysis was performed to investigate the differentially expressed genes between normal fibroblasts (NFs) and CAFs, and we identified that the expression of periostin (POSTN), which is known to be overexpressed in various solid tumors and promote cancer progression, was significantly higher in CAFs than in NFs. POSTN increased cell proliferation via NSCLC cells' ERK pathway activation and induced epithelial-mesenchymal transition (EMT), which improved migration in vitro. In addition, POSTN knockdown in CAFs suppressed these effects, and in vivo experiments demonstrated that the POSTN knockdown improved the sensitivity of EGFR-mutant NSCLC cells for osimertinib treatment. Collectively, our results showed that CAF-derived POSTN is involved in tumor growth, migration, EMT induction, and drug resistance in NSCLC. Targeting CAF-secreted POSTN could be a potential therapeutic strategy for NSCLC. KEY MESSAGES: • POSTN is significantly upregulated in CAFs compared to normal fibroblasts in NCSLC. • POSTN increases cell proliferation via activation of the NSCLC cells' ERK pathway. • POSTN induces EMT in NSCLC cells and improves the migration ability. • POSTN knockdown improves the sensitivity for osimertinib in EGFR-mutant NSCLC cells.

DOI： 10.1007/s00109-023-02384-7

Phosphorylated SARM1 is involved in the pathological process of rotenone-induced neurodegeneration. 査読国際誌

Hitoshi Murata, May Tha Zin Phoo, Toshiki Ochi, Nahoko Tomonobu, Ken-Ichi Yamamoto, Rie Kinoshita, Ikuko Miyazaki, Masahiro Nishibori, Masato Asanuma, Masakiyo Sakaguchi

Journal of biochemistry 174 ( 6 ) 533 - 548 2023年11月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Sterile alpha and Toll/interleukin receptor motif-containing protein 1 (SARM1) is a NAD+ hydrolase that plays a key role in axonal degeneration and neuronal cell death. We reported that c-Jun N-terminal kinase (JNK) activates SARM1 through phosphorylation at Ser-548. The importance of SARM1 phosphorylation in the pathological process of Parkinson's disease (PD) has not been determined. We thus conducted the present study by using rotenone (an inducer of PD-like pathology) and neurons derived from induced pluripotent stem cells (iPSCs) from healthy donors and a patient with familial PD PARK2 (FPD2). The results showed that compared to the healthy neurons, FPD2 neurons were more vulnerable to rotenone-induced stress and had higher levels of SARM1 phosphorylation. Similar cellular events were obtained when we used PARK2-knockdown neurons derived from healthy donor iPSCs. These events in both types of PD-model neurons were suppressed in neurons treated with JNK inhibitors, Ca2+-signal inhibitors, or by a SARM1-knockdown procedure. The degenerative events were enhanced in neurons overexpressing wild-type SARM1 and conversely suppressed in neurons overexpressing the SARM1-S548A mutant. We also detected elevated SARM1 phosphorylation in the midbrain of PD-model mice. The results indicate that phosphorylated SARM1 plays an important role in the pathological process of rotenone-induced neurodegeneration.

DOI： 10.1093/jb/mvad068

STAT1/3 signaling suppresses axon degeneration and neuronal cell death through regulation of NAD+-biosynthetic and consuming enzymes. 査読国際誌

Hitoshi Murata, Yu Yasui, Kazuma Oiso, Toshiki Ochi, Nahoko Tomonobu, Ken-Ichi Yamamoto, Rie Kinoshita, Masakiyo Sakaguchi

Cellular signalling 108 110717 - 110717 2023年8月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Nicotinamide adenine dinucleotide (NAD)+-biosynthetic and consuming enzymes are involved in various intracellular events through the regulation of NAD+ metabolism. Recently, it has become clear that alterations in the expression of NAD+-biosynthetic and consuming enzymes contribute to the axonal stability of neurons. We explored soluble bioactive factor(s) that alter the expression of NAD+-metabolizing enzymes and found that cytokine interferon (IFN)-γ increased the expression of nicotinamide nucleotide adenylyltransferase 2 (NMNAT2), an NAD+-biosynthetic enzyme. IFN-γ activated signal transducers and activators of transcription 1 and 3 (STAT1/3) followed by c-Jun N-terminal kinase (JNK) suppression. As a result, STAT1/3 increased the expression of NMNAT2 at both mRNA and protein levels in a dose- and time-dependent manner and, at the same time, suppressed activation of sterile alpha and Toll/interleukin receptor motif-containing 1 (SARM1), an NAD+-consuming enzyme, and increased intracellular NAD+ levels. We examined the protective effect of STAT1/3 signaling against vincristine-mediated cell injury as a model of chemotherapy-induced peripheral neuropathy (CIPN), in which axonal degeneration is involved in disease progression. We found that IFN-γ-mediated STAT1/3 activation inhibited vincristine-induced downregulation of NMNAT2 and upregulation of SARM1 phosphorylation, resulting in modest suppression of subsequent neurite degradation and cell death. These results indicate that STAT1/3 signaling induces NMNAT2 expression while simultaneously suppressing SARM1 phosphorylation, and that both these actions contribute to suppression of axonal degeneration and cell death.

DOI： 10.1016/j.cellsig.2023.110717

PPARα activation partially drives NAFLD development in liver-specific Hnf4a-null mice. 査読国際誌

Carlos Ichiro Kasano-Camones, Masayuki Takizawa, Noriyasu Ohshima, Chinatsu Saito, Wakana Iwasaki, Yuko Nakagawa, Yoshio Fujitani, Ryo Yoshida, Yoshifumi Saito, Takashi Izumi, Shin-Ichi Terawaki, Masakiyo Sakaguchi, Frank J Gonzalez, Yusuke Inoue

Journal of biochemistry 173 ( 5 ) 393 - 411 2023年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Oxford University Press (OUP)

Abstract

HNF4α regulates various genes to maintain liver function. There have been reports linking HNF4α expression to the development of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). In this study, liver-specific Hnf4a-deficient mice (Hnf4aΔHep mice) developed hepatosteatosis and liver fibrosis, and they were found to have difficulty utilizing glucose. In Hnf4aΔHep mice, the expression of fatty acid oxidation-related genes, which are PPARα target genes, was increased in contrast to the decreased expression of PPARα, suggesting that Hnf4aΔHep mice take up more lipids in the liver instead of glucose. Furthermore, Hnf4aΔHep/Ppara-/- mice, which are simultaneously deficient in HNF4α and PPARα, showed improved hepatosteatosis and fibrosis. Increased C18:1 and C18:1/C18:0 ratio was observed in the livers of Hnf4aΔHep mice and the transactivation of PPARα target gene was induced by C18:1. When the C18:1/C18:0 ratio was close to that of Hnf4aΔHep mouse liver, a significant increase in transactivation was observed. In addition, the expression of Pgc1a, a coactivator of PPARs, was increased, suggesting that elevated C18:1 and Pgc1a expression could contribute to PPARα activation in Hnf4aΔHep mice. These insights may contribute to the development of new diagnostic and therapeutic approaches for NAFLD by focusing on the HNF4α and PPARα signaling cascade.

DOI： 10.1093/jb/mvad005

Novel extracellular role of REIC/Dkk-3 protein in PD-L1 regulation in cancer cells. 査読国際共著国際誌

Yuma Gohara, Nahoko Tomonobu, Rie Kinoshita, Junichiro Futami, Léna Audebert, Youyi Chen, Ni Luh Gede Yoni Komalasari, Fan Jiang, Chikako Yoshizawa, Hitoshi Murata, Ken-Ichi Yamamoto, Masami Watanabe, Hiromi Kumon, Masakiyo Sakaguchi

Journal of molecular medicine (Berlin, Germany) 101 ( 4 ) 431 - 447 2023年4月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The adenovirus-REIC/Dkk-3 expression vector (Ad-REIC) has been the focus of numerous clinical studies due to its potential for the quenching of cancers. The cancer-suppressing mechanisms of the REIC/DKK-3 gene depend on multiple pathways that exert both direct and indirect effects on cancers. The direct effect is triggered by REIC/Dkk-3-mediated ER stress that causes cancer-selective apoptosis, and the indirect effect can be classified in two ways: (i) induction, by Ad-REIC-mis-infected cancer-associated fibroblasts, of the production of IL-7, an important activator of T cells and NK cells, and (ii) promotion, by the secretory REIC/Dkk-3 protein, of dendritic cell polarization from monocytes. These unique features allow Ad-REIC to exert effective and selective cancer-preventative effects in the manner of an anticancer vaccine. However, the question of how the REIC/Dkk-3 protein leverages anticancer immunity has remained to be answered. We herein report a novel function of the extracellular REIC/Dkk-3-namely, regulation of an immune checkpoint via modulation of PD-L1 on the cancer-cell surface. First, we identified novel interactions of REIC/Dkk-3 with the membrane proteins C5aR, CXCR2, CXCR6, and CMTM6. These proteins all functioned to stabilize PD-L1 on the cell surface. Due to the dominant expression of CMTM6 among the proteins in cancer cells, we next focused on CMTM6 and observed that REIC/Dkk-3 competed with CMTM6 for PD-L1, thereby liberating PD-L1 from its complexation with CMTM6. The released PD-L1 immediately underwent endocytosis-mediated degradation. These results will enhance our understanding of not only the physiological nature of the extracellular REIC/Dkk-3 protein but also the Ad-REIC-mediated anticancer effects. KEY MESSAGES: • REIC/Dkk-3 protein effectively suppresses breast cancer progression through an acceleration of PD-L1 degradation. • PD-L1 stability on the cancer cell membrane is kept high by binding with mainly CMTM6. • Competitive binding of REIC/Dkk-3 protein with CMTM6 liberates PD-L1, leading to PD-L1 degradation.

DOI： 10.1007/s00109-023-02292-w

SPRED2: A Novel Regulator of Epithelial-Mesenchymal Transition and Stemness in Hepatocellular Carcinoma Cells. 査読国際誌

Tong Gao, Xu Yang, Masayoshi Fujisawa, Toshiaki Ohara, Tianyi Wang, Nahoko Tomonobu, Masakiyo Sakaguchi, Teizo Yoshimura, Akihiro Matsukawa

International journal of molecular sciences 24 ( 5 ) 2023年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

The downregulation of SPRED2, a negative regulator of the ERK1/2 pathway, was previously detected in human cancers; however, the biological consequence remains unknown. Here, we investigated the effects of SPRED2 loss on hepatocellular carcinoma (HCC) cell function. Human HCC cell lines, expressing various levels of SPRED2 and SPRED2 knockdown, increased ERK1/2 activation. SPRED2-knockout (KO)-HepG2 cells displayed an elongated spindle shape with increased cell migration/invasion and cadherin switching, with features of epithelial-mesenchymal transition (EMT). SPRED2-KO cells demonstrated a higher ability to form spheres and colonies, expressed higher levels of stemness markers and were more resistant to cisplatin. Interestingly, SPRED2-KO cells also expressed higher levels of the stem cell surface markers CD44 and CD90. When CD44+CD90+ and CD44-CD90- populations from WT cells were analyzed, a lower level of SPRED2 and higher levels of stem cell markers were detected in CD44+CD90+ cells. Further, endogenous SPRED2 expression decreased when WT cells were cultured in 3D, but was restored in 2D culture. Finally, the levels of SPRED2 in clinical HCC tissues were significantly lower than those in adjacent non-HCC tissues and were negatively associated with progression-free survival. Thus, the downregulation of SPRED2 in HCC promotes EMT and stemness through the activation of the ERK1/2 pathway, and leads to more malignant phenotypes.

DOI： 10.3390/ijms24054996

LOXL1 and LOXL4 are novel target genes of the Zn2+-bound form of ZEB1 and play a crucial role in the acceleration of invasive events in triple-negative breast cancer cells. 査読国際共著国際誌

Daisuke Hirabayashi, Ken-Ichi Yamamoto, Akihiro Maruyama, Nahoko Tomonobu, Rie Kinoshita, Youyi Chen, Ni Luh Gede Yoni Komalasari, Hitoshi Murata, Yuma Gohara, Fan Jiang, Jin Zhou, I Made Winarsa Ruma, I Wayan Sumardika, Akira Yamauchi, Futoshi Kuribayashi, Shinichi Toyooka, Yusuke Inoue, Masakiyo Sakaguchi

Frontiers in oncology 13 1142886 - 1142886 2023年2月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers Media SA

Background

EMT has been proposed to be a crucial early event in cancer metastasis. EMT is rigidly regulated by the action of several EMT-core transcription factors, particularly ZEB1. We previously revealed an unusual role of ZEB1 in the S100A8/A9-mediated metastasis in breast cancer cells that expressed ZEB1 at a significant level and showed that the ZEB1 was activated on the MCAM-downstream pathway upon S100A8/A9 binding. ZEB1 is well known to require Zn²⁺ for its activation based on the presence of several Zn-finger motifs in the transcription factor. However, how Zn²⁺-binding works on the pleiotropic role of ZEB1 through cancer progression has not been fully elucidated.

Methods

We established the engineered cells, MDA-MB-231 MutZEB1 (MDA-MutZEB1), that stably express MutZEB1 (ΔZn). The cells were then evaluated in vitro for their invasion activities. Finally, an RNA-Seq analysis was performed to compare the gene alteration profiles of the established cells comprehensively.

Results

MDA-MutZEB1 showed a significant loss of the EMT, ultimately stalling the invasion. Inclusive analysis of the transcription changes after the expression of MutZEB1 (ΔZn) in MDA-MB-231 cells revealed the significant downregulation of LOX family genes, which are known to play a critical role in cancer metastasis. We found that LOXL1 and LOXL4 remarkably enhanced cancer invasiveness among the LOX family genes with altered expression.

Conclusions

These findings indicate that ZEB1 potentiates Zn²⁺-mediated transcription of plural EMT-relevant factors, including LOXL1 and LOXL4, whose upregulation plays a critical role in the invasive dissemination of breast cancer cells.

DOI： 10.3389/fonc.2023.1142886

Lysyl oxidase-like 4 exerts an atypical role in breast cancer progression that is dependent on the enzymatic activity that targets the cell-surface annexin A2. 査読国際共著国際誌

Ni Luh Gede Yoni Komalasari, Nahoko Tomonobu, Rie Kinoshita, Youyi Chen, Yoshihiko Sakaguchi, Yuma Gohara, Fan Jiang, Ken-Ich Yamamoto, Hitoshi Murata, I Made Winarsa Ruma, I Wayan Sumardika, Jin Zhou, Akira Yamauchi, Futoshi Kuribayashi, Yusuke Inoue, Shinichi Toyooka, Masakiyo Sakaguchi

Frontiers in oncology 13 1142907 - 1142907 2023年

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: LOX family members are reported to play pivotal roles in cancer. Unlike their enzymatic activities in collagen cross-linking, their precise cancer functions are unclear. We revealed that LOXL4 is highly upregulated in breast cancer cells, and we thus sought to define an unidentified role of LOXL4 in breast cancer. METHODS: We established the MDA-MB-231 sublines MDA-MB-231-LOXL4 mutCA and -LOXL4 KO, which stably overexpress mutant LOXL4 that loses its catalytic activity and genetically ablates the intrinsic LOXL4 gene, respectively. In vitro and in vivo evaluations of these cells' activities of cancer outgrowth were conducted by cell-based assays in cultures and an orthotopic xenograft model, respectively. The new target (s) of LOXL4 were explored by the MS/MS analytic approach. RESULTS: Our in vitro results revealed that both the overexpression of mutCA and the KO of LOXL4 in cells resulted in a marked reduction of cell growth and invasion. Interestingly, the lowered cellular activities observed in the engineered cells were also reflected in the mouse model. We identified a novel binding partner of LOXL4, i.e., annexin A2. LOXL4 catalyzes cell surface annexin A2 to achieve a cross-linked multimerization of annexin A2, which in turn prevents the internalization of integrin β-1, resulting in the locking of integrin β-1 on the cell surface. These events enhance the promotion of cancer cell outgrowth. CONCLUSIONS: LOXL4 has a new role in breast cancer progression that occurs via an interaction with annexin A2 and integrin β-1 on the cell surface.

DOI： 10.3389/fonc.2023.1142907

Toll-like receptor 4 promotes bladder cancer progression upon S100A8/A9 binding, which requires TIRAP-mediated TPL2 activation. 査読国際誌

Acosta Gonzalez Herik Rodrigo, Nahoko Tomonobu, Haruka Yoneda, Rie Kinoshita, Yosuke Mitsui, Takuya Sadahira, Shin-Ichi Terawaki, Yuma Gohara, Ni Luh Gede Yoni Komalasari, Fan Jiang, Hitoshi Murata, Ken-Ichi Yamamoto, Junichiro Futami, Akira Yamauchi, Futoshi Kuribayashi, Yusuke Inoue, Eisaku Kondo, Shinichi Toyooka, Masahiro Nishibori, Masami Watanabe, Yasutomo Nasu, Masakiyo Sakaguchi

Biochemical and biophysical research communications 634 83 - 91 2022年12月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Bladder cancer is an often widely disseminated and deadly cancer. To block the malignant outgrowth of bladder cancer, we must elucidate the molecular-level characteristics of not only bladder cancer cells but also their surrounding milieu. As part of this effort, we have long been studying extracellular S100A8/A9, which is elevated by the inflammation associated with certain cancers. Extracellularly enriched S100A8/A9 can hasten a shift to metastatic transition in multiple types of cancer cells. Intriguingly, high-level S100A8/A9 has been detected in the urine of bladder-cancer patients, and the level increases with the stage of malignancy. Nonetheless, S100A8/A9 has been investigated mainly as a potential biomarker of bladder cancers, and there have been no investigations of its role in bladder-cancer growth and metastasis. We herein report that extracellular S100A8/A9 induces upregulation of growth, migration and invasion in bladder cancer cells through its binding with cell-surface Toll-like receptor 4 (TLR4). Our molecular analysis revealed the TLR4 downstream signal that accelerates such cancer cell events. Tumor progression locus 2 (TPL2) was a key factor facilitating the aggressiveness of cancer cells. Upon binding of S100A8/A9 with TLR4, TPL2 activation was enhanced by an action with a TLR4 adaptor molecule, TIR domain-containing adaptor protein (TIRAP), which in turn led to activation of the mitogen-activated protein kinase (MAPK) cascade of TPL2. Finally, we showed that sustained inhibition of TLR4 in cancer cells effectively dampened cancer survival in vivo. Collectively, our results indicate that the S100A8/A9-TLR4-TPL2 axis influences the growth, survival, and invasive motility of bladder cancer cells.

DOI： 10.1016/j.bbrc.2022.09.116

Inhibiting S100A8/A9 attenuates airway obstruction in a mouse model of heterotopic tracheal transplantation. 査読国際誌

Dai Shimizu, Mikio Okazaki, Seiichiro Sugimoto, Rie Kinoshita, Kentaro Nakata, Shin Tanaka, Kohei Hashimoto, Kentaroh Miyoshi, Masaomi Yamane, Akihiro Matsukawa, Masakiyo Sakaguchi, Shinichi Toyooka

Biochemical and biophysical research communications 629 86 - 94 2022年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Although bronchiolitis obliterans syndrome (BOS) is a major cause of death after lung transplantation, an effective drug therapy for BOS has not yet developed. Here, we assessed the effectiveness of a neutralizing anti-S100 calcium binding protein (S100) A8/A9 antibody against BOS. A murine model of heterotopic tracheal transplantation was used. Mice were intraperitoneally administered control IgG or the S100A8/A9 antibody on day 0 and twice per week until they were sacrificed. Tissue sections were used to evaluate the obstruction ratio, epithelium-preservation ratio, α-smooth muscle actin (SMA)-positive myofibroblast infiltration, and luminal cell death. Quantitative reverse transcriptase-polymerase chain reaction analysis was performed to analyze the mRNA-expression levels of collagen, inflammatory cytokines, and chemokines on days 7, 14, and 21. The anti-S100A8/A9 antibody significantly improved the obstruction ratio and epithelium-preservation ratio, with less α-SMA-positive myofibroblast infiltration compared to the control group. Antibody treatment reduced the type-III collagen: type-I collagen gene-expression ratio. The antibody also significantly suppressed the number of dead cells in the graft lumen. The expression levels of tumor growth factor β1 and C-C motif chemokine 2 on day 21, but not those of interleukin-1β, interleukin-6, and tumor necrosis factor α, were significantly suppressed by S100A8/A9 antibody treatment. These findings suggest that S100A8/A9 may be a potential therapeutic target for BOS after lung transplantation.

DOI： 10.1016/j.bbrc.2022.08.087

Functional Blockage of S100A8/A9 Ameliorates Ischemia-Reperfusion Injury in the Lung. 査読国際誌

Kentaro Nakata, Mikio Okazaki, Tomohisa Sakaue, Rie Kinoshita, Yuhei Komoda, Dai Shimizu, Haruchika Yamamoto, Shin Tanaka, Ken Suzawa, Kazuhiko Shien, Kentaroh Miyoshi, Hiromasa Yamamoto, Toshiaki Ohara, Seiichiro Sugimoto, Masaomi Yamane, Akihiro Matsukawa, Masakiyo Sakaguchi, Shinichi Toyooka

Bioengineering (Basel, Switzerland) 9 ( 11 ) 2022年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

(1) Background: Lung ischemia-reperfusion (IR) injury increases the mortality and morbidity of patients undergoing lung transplantation. The objective of this study was to identify the key initiator of lung IR injury and to evaluate pharmacological therapeutic approaches using a functional inhibitor against the identified molecule. (2) Methods: Using a mouse hilar clamp model, the combination of RNA sequencing and histological investigations revealed that neutrophil-derived S100A8/A9 plays a central role in inflammatory reactions during lung IR injury. Mice were assigned to sham and IR groups with or without the injection of anti-S100A8/A9 neutralizing monoclonal antibody (mAb). (3) Results: Anti-S100A8/A9 mAb treatment significantly attenuated plasma S100A8/A9 levels compared with control IgG. As evaluated by oxygenation capacity and neutrophil infiltration, the antibody treatment dramatically ameliorated the IR injury. The gene expression levels of cytokines and chemokines induced by IR injury were significantly reduced by the neutralizing antibody. Furthermore, the antibody treatment significantly reduced TUNEL-positive cells, indicating the presence of apoptotic cells. (4) Conclusions: We identified S100A8/A9 as a novel therapeutic target against lung IR injury.

DOI： 10.3390/bioengineering9110673

Inhibition of pancreatic cancer-cell growth and metastasis in vivo by a pyrazole compound characterized as a cell-migration inhibitor by an in vitro chemotaxis assay. 査読国際誌

Shuichiro Okamoto, Kei Miyano, Tominari Choshi, Norihiko Sugisawa, Takashi Nishiyama, Rika Kotouge, Masahiro Yamamura, Masakiyo Sakaguchi, Rie Kinoshita, Nahoko Tomonobu, Naoki Katase, Kyo Sasaki, Sohji Nishina, Keisuke Hino, Koji Kurose, Mikio Oka, Hisako Kubota, Tomio Ueno, Toshihiro Hirai, Hideyo Fujiwara, Chikage Kawai, Masumi Itadani, Aya Morihara, Kouji Matsushima, Shiro Kanegasaki, Robert M Hoffman, Akira Yamauchi, Futoshi Kuribayashi

Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 155 113733 - 113733 2022年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Pancreatic cancer is recalcitrant to treatment as it is highly metastatic and rapidly progressive. While observing the behavior of human pancreatic BxPC-3 cells using an optical assay device called TAXIScan, we found that several synthetic pyrazole and pyrimidine derivatives inhibited cell migration. One such compound, 14-100, inhibited metastasis of fluorescence-labeled BxPC-3 cells, which were transplanted into the pancreas of nude mice as a subcutaneously grown cancer fragment. Surprisingly, despite its low cytotoxicity, the compound also showed an inhibitory effect on cancer cell proliferation in vivo, suggesting that the compound alters cancer cell characteristics needed to grow in situ. Single-cell RNA-sequencing revealed changes in gene expression associated with metastasis, angiogenesis, inflammation, and epithelial-mesenchymal transition. These data suggest that the compound 14-100 could be a good drug candidate against pancreatic cancer.

DOI： 10.1016/j.biopha.2022.113733

Exosomal Wnt7a from a low metastatic subclone promotes lung metastasis of a highly metastatic subclone in the murine 4t1 breast cancer. 査読国際誌

Chunning Li, Teizo Yoshimura, Miao Tian, Yuze Wang, Takamasa Kondo, Ken-Ichi Yamamoto, Masayoshi Fujisawa, Toshiaki Ohara, Masakiyo Sakaguchi, Akihiro Matsukawa

Breast cancer research : BCR 24 ( 1 ) 60 - 60 2022年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: Patients with triple-negative breast cancer (TNBC) often have poorer prognosis than those with other subtypes because of its aggressive behaviors. Cancer cells are heterogeneous, and only a few highly metastatic subclones metastasize. Although the majority of subclones may not metastasize, they could contribute by releasing factors that increase the capacity of highly metastatic cells and/or provide a favorable tumor microenvironment (TME). Here, we analyzed the interclonal communication in TNBC which leads to efficient cancer progression, particularly lung metastasis, using the polyclonal murine 4T1 BC model. METHODS: We isolated two 4T1 subclones, LM.4T1 and HM.4T1 cells with a low and a high metastatic potential, respectively, and examined the effects of LM.4T1 cells on the behaviors of HM.4T1 cells using the cell scratch assay, sphere-forming assay, sphere invasion assay, RT-qPCR, and western blotting in vitro. We also examined the contribution of LM.4T1 cells to the lung metastasis of HM.4T1 cells and TME in vivo. To identify a critical factor which may be responsible for the effects by LM.4T1 cells, we analyzed the data obtained from the GEO database. RESULTS: Co-injection of LM.4T1 cells significantly augmented lung metastases by HM.4T1 cells. LM.4T1-derived exosomes promoted the migration and invasion of HM.4T1 cells in vitro, and blocking the secretion of exosome abrogated their effects on HM.4T1 cells. Analyses of data obtained from the GEO database suggested that Wnt7a might be a critical factor responsible for the enhancing effects. In fact, a higher level of Wnt7a was detected in LM.4T1 cells, especially in exosomes, than in HM.4T1 cells, and deletion of Wnt7a in LM.4T1 cells significantly decreased the lung metastasis of HM.4T1 cells. Further, treatment with Wnt7a increased the spheroid formation by HM.4T1 cells via activation of the PI3K/Akt/mTOR signaling pathway. Finally, infiltration of αSMA-positive fibroblasts and angiogenesis was more prominent in tumors of LM.4T1 cells and deletion of Wnt7a in LM.4T1 cells markedly reduced angiogenesis. CONCLUSIONS: We demonstrated, for the first time, that a low metastatic subclone can enhance lung metastasis of highly metastatic subclone via exosomal Wnt7a and propose Wnt7a as a molecular target to treat TNBC patients.

DOI： 10.1186/s13058-022-01557-5

Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells. 査読国際共著国際誌

Nahoko Tomonobu, Rie Kinoshita, Hidenori Wake, Yusuke Inoue, I Made Winarsa Ruma, Ken Suzawa, Yuma Gohara, Ni Luh Gede Yoni Komalasari, Fan Jiang, Hitoshi Murata, Ken-Ichi Yamamoto, I Wayan Sumardika, Youyi Chen, Junichiro Futami, Akira Yamauchi, Futoshi Kuribayashi, Eisaku Kondo, Shinichi Toyooka, Masahiro Nishibori, Masakiyo Sakaguchi

International journal of molecular sciences 23 ( 18 ) 10300 - 10300 2022年9月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis.

DOI： 10.3390/ijms231810300

Dkk3/REIC Deficiency Impairs Spermiation, Sperm Fibrous Sheath Integrity and the Sperm Motility of Mice. 査読国際誌

Ruizhi Xue, Wenfeng Lin, Hirofumi Fujita, Jingkai Sun, Rie Kinoshita, Kazuhiko Ochiai, Junichiro Futami, Masami Watanabe, Hideyo Ohuchi, Masakiyo Sakaguchi, Zhengyan Tang, Peng Huang, Yasutomo Nasu, Hiromi Kumon

Genes 13 ( 2 ) 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

The role of Dickkopf-3 (Dkk3)/REIC (The Reduced Expression in Immortalized Cells), a Wnt-signaling inhibitor, in male reproductive physiology remains unknown thus far. To explore the functional details of Dkk3/REIC in the male reproductive process, we studied the Dkk3/REIC knock-out (KO) mouse model. By examining testicular sections and investigating the sperm characteristics (count, vitality and motility) and ultrastructure, we compared the reproductive features between Dkk3/REIC-KO and wild-type (WT) male mice. To further explore the underlying molecular mechanism, we performed RNA sequencing (RNA-seq) analysis of testicular tissues. Our results showed that spermiation failure existed in seminiferous tubules of Dkk3/REIC-KO mice, and sperm from Dkk3/REIC-KO mice exhibited inferior motility (44.09 ± 8.12% vs. 23.26 ± 10.02%, p < 0.01). The Ultrastructure examination revealed defects in the sperm fibrous sheath of KO mice. Although the average count of Dkk3/REIC-KO epididymal sperm was less than that of the wild-types (9.30 ± 0.69 vs. 8.27 ± 0.87, ×106), neither the gap (p > 0.05) nor the difference in the sperm vitality rate (72.83 ± 1.55% vs. 72.50 ± 0.71%, p > 0.05) were statistically significant. The RNA-seq and GO (Gene Oncology) enrichment results indicated that the differential genes were significantly enriched in the GO terms of cytoskeleton function, cAMP signaling and calcium ion binding. Collectively, our research demonstrates that Dkk3/REIC is involved in the process of spermiation, fibrous sheath integrity maintenance and sperm motility of mice.

DOI： 10.3390/genes13020285

Multifaceted Analysis of IL-23A- and/or EBI3-Including Cytokines Produced by Psoriatic Keratinocytes. 査読国際誌

Kota Tachibana, Nina Tang, Hitoshi Urakami, Ai Kajita, Mina Kobashi, Hayato Nomura, Minori Sasakura, Satoru Sugihara, Fan Jiang, Nahoko Tomonobu, Masakiyo Sakaguchi, Mamoru Ouchida, Shin Morizane

International journal of molecular sciences 22 ( 23 ) 2021年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Interleukin (IL) 23 (p19/p40) plays a critical role in the pathogenesis of psoriasis and is upregulated in psoriasis skin lesions. In clinical practice, anti-IL-23Ap19 antibodies are highly effective against psoriasis. IL-39 (p19/ Epstein-Barr virus-induced (EBI) 3), a newly discovered cytokine in 2015, shares the p19 subunit with IL-23. Anti-IL-23Ap19 antibodies may bind to IL-39; also, the cytokine may contribute to the pathogenesis of psoriasis. To investigate IL23Ap19- and/or EBI3-including cytokines in psoriatic keratinocytes, we analyzed IL-23Ap19 and EBI3 expressions in psoriasis skin lesions, using immunohistochemistry and normal human epidermal keratinocytes (NHEKs) stimulated with inflammatory cytokines, using quantitative real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-electrospray tandem mass spectrometry (LC-Ms/Ms). Immunohistochemical analysis showed that IL-23Ap19 and EBI3 expressions were upregulated in the psoriasis skin lesions. In vitro, these expressions were synergistically induced by the triple combination of tumor necrosis factor (TNF)-α, IL-17A, and interferon (IFN)-γ, and suppressed by dexamethasone, vitamin D3, and acitretin. In ELISA and LC-Ms/Ms analyses, keratinocyte-derived IL-23Ap19 and EBI3, but not heterodimeric forms, were detected with humanized anti-IL-23Ap19 monoclonal antibodies, tildrakizumab, and anti-EBI3 antibodies, respectively. Psoriatic keratinocytes may express IL-23Ap19 and EBI3 proteins in a monomer or homopolymer, such as homodimer or homotrimer.

DOI： 10.3390/ijms222312659

Oncogenic potential of human pluripotent stem cell-derived lung organoids with HER2 overexpression. 査読国際誌

Akihiro Miura, Daisuke Yamada, Masahiro Nakamura, Shuta Tomida, Dai Shimizu, Yan Jiang, Tomoka Takao, Hiromasa Yamamoto, Ken Suzawa, Kazuhiko Shien, Masaomi Yamane, Masakiyo Sakaguchi, Shinichi Toyooka, Takeshi Takarada

International journal of cancer 149 ( 8 ) 1593 - 1604 2021年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Lung adenocarcinoma (LUAD) is the most common types among lung cancers generally arising from terminal airway and understanding of multistep carcinogenesis is crucial to develop novel therapeutic strategy for LUAD. Here we used human induced pluripotent stem cells (hiPSCs) to establish iHER2-hiPSCs in which doxycycline induced the expression of the oncoprotein human epidermal growth factor receptor 2 (HER2)/ERBB2. Lung progenitors that differentiated from iHER2-hiPSCs, which expressed NKX2-1/TTF-1 known as a lung lineage maker, were cocultured with human fetal fibroblast and formed human lung organoids (HLOs) comprising alveolar type 2-like cells. HLOs that overexpressed HER2 transformed to tumor-like structures similar to atypical adenomatous hyperplasia, which is known for lung precancerous lesion and upregulated the activities of oncogenic signaling cascades such as RAS/RAF/MAPK and PI3K/AKT/mTOR. The degree of morphological irregularity and proliferation capacity were significantly higher in HLOs from iHER2-hiPSCs. Moreover, the transcriptome profile of the HLOs shifted from a normal lung tissue-like state to one characteristic of clinical LUAD with HER2 amplification. Our results suggest that hiPSC-derived HLOs may serve as a model to recapitulate the early tumorigenesis of LUAD and would provide new insights into the molecular basis of tumor initiation and progression.

DOI： 10.1002/ijc.33713

マウス異所性気管移植モデルを用いたS100A8/A9抗体の慢性移植肺機能不全に対する効果の検討

清水大, 岡崎幹生, 木下理恵, 中田憲太郎, 三好健太郎, 大谷真二, 杉本誠一郎, 山根正修, 阪口政清, 豊岡伸一

移植 56 ( 総会臨時 ) 369 - 369 2021年9月

Inhibitory effects of RAGE-aptamer on development of monocrotaline-induced pulmonary arterial hypertension in rats. 査読国際誌

Kazufumi Nakamura, Satoshi Akagi, Kentaro Ejiri, Masashi Yoshida, Toru Miyoshi, Masakiyo Sakaguchi, Naofumi Amioka, Luh Oliva Saraswati Suastika, Megumi Kondo, Rie Nakayama, Yoichi Takaya, Yuichiro Higashimoto, Kei Fukami, Hiromi Matsubara, Hiroshi Ito

Journal of cardiology 78 ( 1 ) 12 - 16 2021年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: The receptor for advanced glycation end products (RAGE), a transmembrane receptor belonging to the immunoglobulin superfamily, is overexpressed in pulmonary artery smooth muscle cells (PASMCs) in patients with pulmonary arterial hypertension (PAH) and is implicated in the etiology of PAH. Recently, we reported that RAGE-aptamer, a short and single-stranded DNA directed against RAGE, inhibited an inappropriate increase in cultured PASMCs in PAH. The aim of this study was to determine the efficacy of RAGE-aptamer in monocrotaline-induced PAH in rats. METHODS AND RESULTS: Rats were assigned to either an untreated control group, a group that received continuous subcutaneous administration of RAGE-aptamer immediately after monocrotaline injection, or a group that received control-aptamer immediately after monocrotaline injection. All rats survived 21 days after injection of monocrotaline and control-aptamer or RAGE-aptamer. Injection of monocrotaline with continuous subcutaneous delivery of control-aptamer resulted in higher right ventricular systolic pressure compared with controls. This increase was attenuated by continuous subcutaneous delivery of RAGE-aptamer. The proportion of small pulmonary arteries with full muscularization was greater in the monocrotaline and control-aptamer group than in the control group. Continuous subcutaneous delivery of RAGE-aptamer significantly reduced the percentage of small pulmonary arteries with full muscularization. CONCLUSIONS: Continuous subcutaneous delivery of RAGE-aptamer suppresses development of monocrotaline-induced PAH in rats. Inhibition of RAGE ameliorates muscularization of small pulmonary arteries. Treatment with RAGE-aptamer might be a new therapeutic option for PAH.

DOI： 10.1016/j.jjcc.2020.12.009

PLOD2-driven IL-6/STAT3 signaling promotes the invasion and metastasis of oral squamous cell carcinoma via activation of integrin β1. 査読国際誌

Ken Saito, Ayaka Mitsui, I Wayan Sumardika, Yusuke Yokoyama, Masakiyo Sakaguchi, Eisaku Kondo

International journal of oncology 58 ( 6 ) 2021年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

We previously reported that high expression of procollagen‑lysine 2‑oxoglutarate 5‑dioxygenase 2 (PLOD2) leads to stabilization and plasma membrane translocation of integrin β1 to promote the invasion and metastasis of oral squamous cell carcinoma (SCC). The present study aimed to further understand the relationship between PLOD2‑integrin β1 signaling and the tumor microenvironment. This study provided further advanced insights indicating that elevated interleukin (IL)‑6 in the tumor microenvironment acts as a key molecule that triggers PLOD2‑integrin β1 axis‑derived acceleration of tumor invasion and metastasis. It was found using the dual‑luciferase reporter assay system that signal transducer and activator of transcription 3 (STAT3) activation by IL‑6 was essential for increasing the expression levels of PLOD2 through direct activation of the PLOD2 promoter in oral SCC, whereas IL‑6 stimulation did not contribute to integrin β1 expression or the subsequent maturation process towards a functional form on the plasma membrane. Furthermore, the expression of IL‑6 in oral SCC tissues was mainly observed in the tumor stroma. Finally, with double immunofluorescence staining, it was found that IL‑6 expression occurred in CD163‑positive M2 macrophages distributed around the tumor nest. These results combined with our previous results indicate that as IL‑6 significantly increases STAT3‑mediated PLOD2 promoter activity, IL‑6 released by M2‑type tumor‑associated macrophages is a crucial factor that promotes PLOD2‑integrin β1 axis‑enhanced invasion and metastasis of oral SCC cells.

DOI： 10.3892/ijo.2021.5209

Presence of Microplastics in Four Types of Shellfish Purchased at Fish Markets in Okayama City, Japan. 査読

Kenichi Yamamoto, Toshiyuki Oshiki, Hiroko Kagawa, Masayoshi Namba, Masakiyo Sakaguchi

Acta medica Okayama 75 ( 3 ) 381 - 384 2021年6月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The worldwide microplastic pollution in our environment is a matter of great concern. Harmful effects of plastics have been reported in various types of organisms including murine animals. We examined the presence of microplastics in four types of shellfish purchased from fish markets in Okayama, Japan and served to the public: short-neck clam (Ruditapes philippinarum, asari in Japanese), hard-shell clam (Meretrix lusoria, hamaguri), brackishwater clam (Cyrenidae, shijimi), and oyster (Crassostrea gigas, kaki). Our analyses demonstrated that approx. 3 pieces of microplastics were present per single shellfish, based on the division of the total number of pieces of microplastic obtained from all 4 types of shellfish by the total number of shellfish examined. Since health problems in humans due to microplastics have not yet been confirmed, further examinations of the effects of ingested microplastics are needed.

DOI： 10.18926/AMO/62234

肺腺癌におけるSpred2の発現と増殖、浸潤との関連の検討

太田陽子, 藤澤真義, 吉村禎造, スマルディカ・イワヤン, 枝園和彦, 阪口政清, 豊岡伸一, 松川昭博

日本病理学会会誌 110 ( 1 ) 291 - 291 2021年3月

異なる亜集団の腫瘍細胞から放出されたエクソソームWnt7aによリ4T1細胞の転移は促進される(4T1 cells promote tumor metastasis by exosomal Wnt7a released from distinct subpopulations)

李春寧, 大原利章, 藤澤真義, 阪口政清, 山本健一, 田ミャオ, 王宇沢, 吉村禎造, 松川昭博

日本病理学会会誌 110 ( 1 ) 231 - 231 2021年3月

Histidine-Rich Glycoprotein Stimulates Human Neutrophil Phagocytosis and Prolongs Survival through CLEC1A. 査読国際誌

Yohei Takahashi, Hidenori Wake, Masakiyo Sakaguchi, Yukinori Yoshii, Kiyoshi Teshigawara, Dengli Wang, Masahiro Nishibori

Journal of immunology (Baltimore, Md. : 1950) 206 ( 4 ) 737 - 750 2021年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Histidine-rich glycoprotein (HRG) is a multifunctional plasma protein and maintains the homeostasis of blood cells and vascular endothelial cells. In the current study, we demonstrate that HRG and recombinant HRG concentration dependently induced the phagocytic activity of isolated human neutrophils against fluorescence-labeled Escherichia coli and Staphylococcus aureus through the stimulation of CLEC1A receptors, maintaining their spherical round shape. The phagocytosis-inducing effects of HRG were inhibited by a specific anti-HRG Ab and enhanced by opsonization of bacteria with diluted serum. HRG and C5a prolonged the survival time of isolated human neutrophils, in association with a reduction in the spontaneous production of extracellular ROS. In contrast, HRG maintained the responsiveness of neutrophils to TNF-α, zymosan, and E. coli with regard to reactive oxygen species production. The blocking Ab for CLEC1A and recombinant CLEC1A-Fc fusion protein significantly inhibited the HRG-induced neutrophil rounding, phagocytic activity, and prolongation of survival time, suggesting the involvement of the CLEC1A receptor in the action of HRG on human neutrophils. These results as a whole indicated that HRG facilitated the clearance of E. coli and S. aureus by maintaining the neutrophil morphology and phagocytosis, contributing to the antiseptic effects of HRG in vivo.

DOI： 10.4049/jimmunol.2000817

The heterodimer S100A8/A9 is a potent therapeutic target for idiopathic pulmonary fibrosis. 査読国際誌

Kota Araki, Rie Kinoshita, Nahoko Tomonobu, Yuma Gohara, Shuta Tomida, Yuta Takahashi, Satoru Senoo, Akihiko Taniguchi, Junko Itano, Ken-Ichi Yamamoto, Hitoshi Murata, Ken Suzawa, Kazuhiko Shien, Hiromasa Yamamoto, Mikio Okazaki, Seiichiro Sugimoto, Kouichi Ichimura, Masahiro Nishibori, Nobuaki Miyahara, Shinichi Toyooka, Masakiyo Sakaguchi

Journal of molecular medicine (Berlin, Germany) 99 ( 1 ) 131 - 145 2021年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

In patients with interstitial pneumonia, pulmonary fibrosis is an irreversible condition that can cause respiratory failure. Novel treatments for pulmonary fibrosis are necessary. Inflammation is thought to activate lung fibroblasts, resulting in pulmonary fibrosis. Of the known inflammatory molecules, we have focused on S100A8/A9 from the onset of inflammation to the subsequent progression of inflammation. Our findings confirmed the high expression of S100A8/A9 in specimens from patients with pulmonary fibrosis. An active role of S100A8/A9 was demonstrated not only in the proliferation of fibroblasts but also in the fibroblasts' differentiation to myofibroblasts (the active form of fibroblasts). S100A8/A9 also forced fibroblasts to upregulate the production of collagen. These effects were induced via the receptor of S100A8/A9, i.e., the receptor for advanced glycation end products (RAGE), on fibroblasts. The anti-S100A8/A9 neutralizing antibody inhibited the effects of S100A8/A9 on fibroblasts and suppressed the progression of fibrosis in bleomycin (BLM)-induced pulmonary fibrosis mouse model. Our findings strongly suggest a crucial role of S100A8/A9 in pulmonary fibrosis and the usefulness of S100A8/A9-targeting therapy for fibrosis interstitial pneumonia. HIGHLIGHTS: S100A8/A9 level is highly upregulated in the IPF patients' lungs as well as the blood. S100A8/A9 promotes not only the growth of fibroblasts but also differentiation to myofibroblasts. The cell surface RAGE acts as a crucial receptor to the extracellular S100A8/A9 in fibroblasts. The anti-S100A8/A9 antibody effectively suppresses the progression of IPF in a mouse model. In idiopathic pulmonary fibrosis (IPF), S100A8/A9, a heterodimer composed of S100A8 and S100A9 proteins, plays a crucial role in the onset of inflammation and the subsequent formation of a feed-forward inflammatory loop that promotes fibrosis. (1) The local, pronounced increase in S100A8/A9 in the injured inflammatory lung region-which is provided mainly by the activated neutrophils and macrophages-exerts strong inflammatory signals accompanied by dozens of inflammatory soluble factors including cytokines, chemokines, and growth factors that further act to produce and secrete S100A8/A9, eventually making a sustainable inflammatory circuit that supplies an indefinite presence of S100A8/A9 in the extracellular space with a mal-increased level. (2) The elevated S100A8/A9 compels fibroblasts to activate through receptor for advanced glycation end products (RAGE), one of the major S100A8/A9 receptors, resulting in the activation of NFκB, leading to fibroblast mal-events (e.g., elevated cell proliferation and transdifferentiation to myofibroblasts) that actively produce not only inflammatory cytokines but also collagen matrices. (3) Finally, the S100A8/A9-derived activation of lung fibroblasts under a chronic inflammation state leads to fibrosis events and constantly worsens fibrosis in the lung. Taken together, these findings suggest that the extracellular S100A8/A9 heterodimer protein is a novel mainstay soluble factor for IPF that exerts many functions as described above (1-3). Against this background, we herein applied the developed S100A8/A9 neutralizing antibody to prevent IPF. The IPF imitating lung fibrosis in an IPF mouse model was effectively blocked by treatment with the antibody, leading to enhanced survival. The developed S100A8/A9 antibody, as an innovative novel biologic, may help shed light on the difficulties encountered with IPF therapy in clinical settings.

DOI： 10.1007/s00109-020-02001-x

Corrigendum: RUNX2 Phosphorylation by Tyrosine Kinase ABL Promotes Breast Cancer Invasion. 査読国際誌

Fang He, Yoshinori Matsumoto, Yosuke Asano, Yuriko Yamamura, Takayuki Katsuyama, Jose La Rose, Nahoko Tomonobu, Ni Luh Gede Yoni Komalasari, Masakiyo Sakaguchi, Robert Rottapel, Jun Wada

Frontiers in oncology 11 729192 - 729192 2021年

記述言語：英語掲載種別：研究論文（学術雑誌）

Activity of transcription factors is normally regulated through interaction with other transcription factors, chromatin remodeling proteins and transcriptional co-activators. In distinction to these well-established transcriptional controls of gene expression, we have uncovered a unique activation model of transcription factors between tyrosine kinase ABL and RUNX2, an osteoblastic master transcription factor, for cancer invasion. We show that ABL directly binds to, phosphorylates, and activates RUNX2 through its SH2 domain in a kinase activity-dependent manner and that the complex formation of these proteins is required for expression of its target gene MMP13. Additionally, we show that the RUNX2 transcriptional activity is dependent on the number of its tyrosine residues that are phosphorylated by ABL. In addition to regulation of RUNX2 activity, we show that ABL transcriptionally enhances RUNX2 expression through activation of the bone morphogenetic protein (BMP)-SMAD pathway. Lastly, we show that ABL expression in highly metastatic breast cancer MDA-MB231 cells is associated with their invasive capacity and that ABL-mediated invasion is abolished by depletion of endogenous RUNX2 or MMP13. Our genetic and biochemical evidence obtained in this study contributes to a mechanistic insight linking ABL-mediated phosphorylation and activation of RUNX2 to induction of MMP13, which underlies a fundamental invasive capacity in cancer and is different from the previously described model of transcriptional activation.

DOI： 10.3389/fonc.2021.729192

Analysis of a plasmid encoding botulinum neurotoxin type G gene in Clostridium argentinense. 査読国際誌

Yoshihiko Sakaguchi, Jumpei Uchiyama, Akira Také, Kazuyoshi Gotoh, Masakiyo Sakaguchi, Tomonori Suzuki, Yumiko Yamamoto, Koji Hosomi, Tomoko Kohda, Masafumi Mukamoto, Shunji Kozaki, Shunji Hayashi, Keiji Oguma

Anaerobe 66 102281 - 102281 2020年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

Clostridium argentinense produces botulinum neurotoxin type G (BoNT/G). We sequenced and analyzed the plasmid harboring the bont/G gene, designated pCAG, in C. argentinense strain 2740. The pCAG consisted of 140,070 bp containing the bont/G gene cluster. Although this gene cluster showed high similarities in its DNA sequence and ORF arrangement to those of other bont gene clusters, the other regions of the plasmid did not. A phylogenetic study suggested that pCAG had a unique evolutionary history compared with other clostridial bont-harboring plasmids. This suggests that pCAG is possibly a novel type of plasmid expressing the bont/G gene in C. argentinense.

DOI： 10.1016/j.anaerobe.2020.102281

Enhancement of intestinal epithelial barrier function by Weissella confusa F213 and Lactobacillus rhamnosus FBB81 probiotic candidates in an in vitro model of hydrogen peroxide-induced inflammatory bowel disease. 査読国際誌

Ni Nengah Dwi Fatmawati, Kazuyoshi Gotoh, I Putu Bayu Mayura, Komang Ayu Nocianitri, Gede Ngurah Rsi Suwardana, Ni Luh Gede Yoni Komalasari, Yan Ramona, Masakiyo Sakaguchi, Osamu Matsushita, I Nengah Sujaya

BMC research notes 13 ( 1 ) 489 - 489 2020年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

OBJECTIVE: Weissella confusa F213 (WCF213) and Lactobacillus rhamnosus FBB81 (LrFBB81) are two probiotic candidates isolated from humans in our previous study. Their functional activity on the mucosal barrier has not yet been adequately investigated. Therefore, the objective of this study was to investigate the effect of these strains on maintaining mucosal integrity in vitro. Caco-2 cell monolayers were pretreated with WCF213 and LrFBB81 before being exposed to hydrogen peroxide. The integrity of mucosal cells was evaluated by measuring the transepithelial resistance (TER), flux of FITC-labelled dextran, and ZO-1 protein distribution with the help of an immunofluorescence method. RESULTS: WCF213 was found to significantly maintain the TER better than the control hydrogen peroxide-treated cells (p < 0.001), followed by the strain combination, and LrFBB81 alone (p < 0.05). The permeability of mucosa was also successfully maintained by the WCF213 strain. This was illustrated by the significant reduction in the flux of FITC-labelled dextran (p < 0.05), which was larger than that exhibited by the other groups. The ZO-1 distribution of strain-treated cells showed less disruption than hydrogen peroxide-treated cells, consistent with the TER and FITC experimental results. These findings indicate that WCF213 and LrFBB81 plays important roles in the maintenance of mucosal integrity in a strain-dependent manner.

DOI： 10.1186/s13104-020-05338-1

Synergistic regulation of hepatic Fsp27b expression by HNF4α and CREBH. 査読国際誌

Carlos Ichiro Kasano-Camones, Masayuki Takizawa, Wakana Iwasaki, Shota Sasaki, Mume Hamada, Aoi Morimoto, Masakiyo Sakaguchi, Frank J Gonzalez, Yusuke Inoue

Biochemical and biophysical research communications 530 ( 2 ) 432 - 439 2020年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

The CIDE (cell death-inducing DFF45-like effector) family composed of CIDEA, CIDEB, CIDEC/FSP27 (fat-specific protein 27), has a critical role in growth of lipid droplets. Of these, CIDEB and CIDEC2/FSP27B are abundant in the liver, and the steatotic livers, respectively. Hepatocyte nuclear factor 4α (HNF4α) has an important role in lipid homeostasis because liver-specific HNF4α-null mice (Hnf4aΔHep mice) exhibit hepatosteatosis. We investigated whether HNF4α directly regulates expression of CIDE family genes. Expression of Cideb and Fsp27b was largely decreased in Hnf4aΔHep mice, while expression of Cidea was increased. Similar results were observed only in CIDEC2, the human orthologue of the Fsp27b, in human hepatoma cell lines in which HNF4α expression was knocked down. Conversely, overexpression of HNF4α strongly induced CIDEC2 expression in hepatoma cell lines. Furthermore, HNF4α transactivated Fsp27b by direct binding to an HNF4α response element in the Fsp27b promoter. In addition, Fsp27b is known to be transactivated by CREBH that is regulated by HNF4α, and expression of CREBH was induced by HNF4α in human hepatoma cells. Co-transfection of HNF4α and CREBH resulted in synergistic transactivation and induction of Fsp27b compared to that of HNF4α or CREBH alone. These results suggest that HNF4α, in conjunction with CREBH, plays an important role in regulation of Fsp27b expression.

DOI： 10.1016/j.bbrc.2020.05.070

Cytotoxic Effects of Alcohol Extracts from a Plastic Wrap (Polyvinylidene Chloride) on Human Cultured Liver Cells and Mouse Primary Cultured Liver Cells. 査読

Ken-Ichi Yamamoto, Hiroko Kagawa, Sakae Arimoto, Xian Wen Tan, Kento Yasui, Toshiyuki Oshiki, Masakiyo Sakaguchi

Acta medica Okayama 74 ( 4 ) 327 - 334 2020年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

An increasing accumulation of microplastics and further degraded nanoplastics in our environment is suspected to have harmful effects on humans and animals. To clarify this problem, we tested the cytotoxicity of two types of plastic wrap on human cultured liver cells and mouse primary cultured liver cells. Alcohol extracts from plastic wrap, i.e., polyvinylidene chloride (PVDC), showed cytotoxic effects on the cells. Alcohol extracts of polyethylene (PE) wrap were not toxic. The commercially available PVDC wrap consists of vinylidene chloride, epoxidized soybean oil, epoxidized linseed oil as a stiffener and stabilizer; we sought to identify which component(s) are toxic. The epoxidized soybean oil and epoxidized linseed oil exerted strong cytotoxicity, but the plastic raw material itself, vinylidene chloride, did not. Our findings indicate that plastic wraps should be used with caution in order to prevent health risks.

DOI： 10.18926/AMO/60371

Neuroplastinβ-mediated upregulation of solute carrier family 22 member 18 antisense (SLC22A18AS) plays a crucial role in the epithelial-mesenchymal transition, leading to lung cancer cells' enhanced motility. 査読国際誌

Karolina Bajkowska, I Wayan Sumardika, Nahoko Tomonobu, Youyi Chen, Ken-Ichi Yamamoto, Rie Kinoshita, Hitoshi Murata, Ni Luh Gede Yoni Komalasari, Fan Jiang, Akira Yamauchi, I Made Winarsa Ruma, Carlos Ichiro Kasano-Camones, Yusuke Inoue, Masakiyo Sakaguchi

Biochemistry and biophysics reports 22 100768 - 100768 2020年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Elsevier BV

Our recent study revealed an important role of the neuroplastin (NPTN)β downstream signal in lung cancer dissemination in the lung. The molecular mechanism of the signal pathway downstream of NPTNβ is a serial activation of the key molecules we identified: tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) adaptor, nuclear factor (NF)IA/NFIB heterodimer transcription factor, and SAM pointed-domain containing ETS transcription factor (SPDEF). The question of how dissemination is controlled by SPDEF under the activated NPTNβ has not been answered. Here, we show that the NPTNβ-SPDEF-mediated induction of solute carrier family 22 member 18 antisense (SLC22A18AS) is definitely required for the epithelial-mesenchymal transition (EMT) through the NPTNβ pathway in lung cancer cells. In vitro, the induced EMT is linked to the acquisition of active cellular motility but not growth, and this is correlated with highly disseminative tumor progression in vivo. The publicly available data also show the poor survival of SLC22A18AS-overexpressing lung cancer patients. Taken together, these data highlight a crucial role of SLC22A18AS in lung cancer dissemination, which provides novel input of this molecule to the signal cascade of NPTNβ. Our findings contribute to a better understanding of NPTNβ-mediated lung cancer metastasis.

DOI： 10.1016/j.bbrep.2020.100768

Histidine-Rich Glycoprotein Inhibits High-Mobility Group Box-1-Mediated Pathways in Vascular Endothelial Cells through CLEC-1A. 査読国際誌

Shangze Gao, Hidenori Wake, Masakiyo Sakaguchi, Dengli Wang, Youhei Takahashi, Kiyoshi Teshigawara, Hui Zhong, Shuji Mori, Keyue Liu, Hideo Takahashi, Masahiro Nishibori

iScience 23 ( 6 ) 101180 - 101180 2020年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

High-mobility group box-1 (HMGB1) protein has been postulated to play a pathogenic role in severe sepsis. Histidine-rich glycoprotein (HRG), a 75 kDa plasma protein, was demonstrated to improve the survival rate of septic mice through the regulation of neutrophils and endothelium barrier function. As the relationship of HRG and HMGB1 remains poorly understood, we investigated the effects of HRG on HMGB1-mediated pathway in endothelial cells, focusing on the involvement of specific receptors for HRG. HRG potently inhibited the HMGB1 mobilization and effectively suppressed rHMGB1-induced inflammatory responses and expression of all three HMGB1 receptors in endothelial cells. Moreover, we first clarified that these protective effects of HRG on endothelial cells were mediated through C-type lectin domain family 1 member A (CLEC-1A) receptor. Thus, current study elucidates protective effects of HRG on vascular endothelial cells through inhibition of HMGB1-mediated pathways may contribute to the therapeutic effects of HRG on severe sepsis.

DOI： 10.1016/j.isci.2020.101180

MAPK Pathway Alterations Correlate with Poor Survival and Drive Resistance to Therapy in Patients with Lung Cancers Driven by ROS1 Fusions. 査読国際誌

Hiroki Sato, Adam J Schoenfeld, Evan Siau, Yue Christine Lu, Huichun Tai, Ken Suzawa, Daisuke Kubota, Allan J W Lui, Besnik Qeriqi, Marissa Mattar, Michael Offin, Masakiyo Sakaguchi, Shinichi Toyooka, Alexander Drilon, Neal X Rosen, Mark G Kris, David Solit, Elisa De Stanchina, Monika A Davare, Gregory J Riely, Marc Ladanyi, Romel Somwar

Clinical cancer research : an official journal of the American Association for Cancer Research 26 ( 12 ) 2932 - 2945 2020年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

PURPOSE: ROS1 tyrosine kinase inhibitors (TKI) provide significant benefit in lung adenocarcinoma patients with ROS1 fusions. However, as observed with all targeted therapies, resistance arises. Detecting mechanisms of acquired resistance (AR) is crucial to finding novel therapies and improve patient outcomes. EXPERIMENTAL DESIGN: ROS1 fusions were expressed in HBEC and NIH-3T3 cells either by cDNA overexpression (CD74/ROS1, SLC34A2/ROS1) or CRISPR-Cas9-mediated genomic engineering (EZR/ROS1). We reviewed targeted large-panel sequencing data (using the MSK-IMPACT assay) patients treated with ROS1 TKIs, and genetic alterations hypothesized to confer AR were modeled in these cell lines. RESULTS: Eight of the 75 patients with a ROS1 fusion had a concurrent MAPK pathway alteration and this correlated with shorter overall survival. In addition, the induction of ROS1 fusions stimulated activation of MEK/ERK signaling with minimal effects on AKT signaling, suggesting the importance of the MAPK pathway in driving ROS1 fusion-positive cancers. Of 8 patients, 2 patients harbored novel in-frame deletions in MEK1 (MEK1delE41_L54) and MEKK1 (MEKK1delH907_C916) that were acquired after ROS1 TKIs, and 2 patients harbored NF1 loss-of-function mutations. Expression of MEK1del or MEKK1del, and knockdown of NF1 in ROS1 fusion-positive cells activated MEK/ERK signaling and conferred resistance to ROS1 TKIs. Combined targeting of ROS1 and MEK inhibited growth of cells expressing both ROS1 fusion and MEK1del. CONCLUSIONS: We demonstrate that downstream activation of the MAPK pathway can mediate of innate acquired resistance to ROS1 TKIs and that patients harboring ROS1 fusion and concurrent downstream MAPK pathway alterations have worse survival. Our findings suggest a treatment strategy to target both aberrations.

DOI： 10.1158/1078-0432.CCR-19-3321

Xylitol acts as an anticancer monosaccharide to induce selective cancer death via regulation of the glutathione level. 査読国際共著国際誌

Nahoko Tomonobu, Ni Luh Gede Yoni Komalasari, I Wayan Sumardika, Fan Jiang, Youyi Chen, Ken-Ichi Yamamoto, Rie Kinoshita, Hitoshi Murata, Yusuke Inoue, Masakiyo Sakaguchi

Chemico-biological interactions 324 109085 - 109085 2020年6月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Herbal medicines and their bioactive compounds are increasingly being recognized as useful drugs for cancer treatments. The parasitic fungus Cordyceps militaris is an attractive anticancer herbal since it shows very powerful anticancer activity due to its phytocompound cordycepin. We previously discovered and reported that a high amount of xylitol is present in Cordyceps militaris extract, and that xylitol unexpectedly showed anticancer activity in a cancer-selective manner. We thus hypothesized that xylitol could become a useful supplement to help prevent various cancers, if we can clarify the specific machinery by which xylitol induces cancer cell death. It is also unclear whether xylitol acts on cancer suppression in vivo as well as in vitro. Here we show for the first time that induction of the glutathione-degrading enzyme CHAC1 is the main cause of xylitol-induced apoptotic cell death in cancer cells. The induction of CHAC1 is required for the endoplasmic reticulum (ER) stress that is triggered by xylitol in cancer cells, and is linked to a second induction of oxidative stress in the treated cells, and eventually leads to apoptotic cell death. Our in vivo approach also demonstrated that an intravenous injection of xylitol had a tumor-suppressing effect in mice, to which the xylitol-triggered ER stress also greatly contributed. We also observed that xylitol efficiently sensitized cancer cells to chemotherapeutic drugs. Based on our findings, a chemotherapeutic strategy combined with xylitol might improve the outcomes of patients facing cancer.

DOI： 10.1016/j.cbi.2020.109085

exMCAM-Fc, an S100A8/A9-mediated-metastasis blocker, efficiently reduced the number of circulating tumor cells that appeared in the blood flow. 査読国際誌

Nahoko Tomonobu, Rie Kinoshita, Masakiyo Sakaguchi

Molecular biology reports 47 ( 6 ) 4879 - 4883 2020年6月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Metastasis is the major cause of treatment failure in cancer patients and of cancer-associated death so that therapeutic regulation of metastasis is very important subject for the cancer treatment. We have been reported that S100A8/A9, a heterodimer complex of S100A8 and S100A9, and its receptors play a crucial role in the lung tropic cancer metastasis, i.e., S100A8/A9 is actively secreted from the lung when cancer mass exists even at remote area from the lung and then functions to attract the distant cancer cells to the lung since cancer cells own the S100A8/A9 receptor(s) on their cell surface. Interestingly, one of the newly developed decoys, exMCAM-Fc, a Fc fusion protein with the extracellular region of melanoma cell adhesion molecule (MCAM), one of the S100A8/A9 receptors, that could prevent the interaction of S100A8/A9 with MCAM, efficiently suppressed the lung tropic cancer metastasis through exerting the several inhibitory effects on the S100A8/A9-mediated cancer cell events including enhanced mobility, invasion and attachment to the endothelial cells. However, it still remains to clarify if the decoy will reduce the number of circulating tumor cells (CTCs) that are defined as substantial cells in the context of organ tropic cancer metastasis. Here, we first show that exMCAM-Fc effectively reduces the number of CTCs in the blood flow of the melanoma bearing mice. The novel finding reinforces the suppressive role of exMCAM-Fc on the cancer metastasis. We therefore expect that exMCAM-Fc may greatly contribute to reduce treatment failure by the efficient blocking of the life threatening cancer metastasis.

DOI： 10.1007/s11033-020-05495-3

Antitumor Effects of Pan-RAF Inhibitor LY3009120 Against Lung Cancer Cells Harboring Oncogenic BRAF Mutation. 査読国際誌

Shunsaku Miyauchi, Kazuhiko Shien, Tatsuaki Takeda, Kota Araki, Kentaro Nakata, Akihiro Miura, Yuta Takahashi, Eisuke Kurihara, Yusuke Ogoshi, Kei Namba, Ken Suzawa, Hiromasa Yamamoto, Mikio Okazaki, Junichi Soh, Shuta Tomida, Masaomi Yamane, Masakiyo Sakaguchi, Shinichi Toyooka

Anticancer research 40 ( 5 ) 2667 - 2673 2020年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND/AIM: The therapeutic strategy for patients with non-small-cell lung cancer (NSCLC) harboring the BRAF non-V600E mutation has not been established. LY3009120, a newly discovered pan-RAF inhibitor, has shown strong antitumor effects in cancers with various BRAF genotypes. This study investigated the antitumor effects of LY3009120 in NSCLC cells harboring the BRAF non-V600E mutation. MATERIALS AND METHODS: We examined the antitumor effects of LY3009120 by MTS assay and flow cytometry. We analyzed the expression status of proteins by western blot. The mouse xenograft models were used for the in vivo experiments. RESULTS: LY3009120 suppressed BRAF-related downstream pathway molecules and induced cleavage of poly ADP-ribose polymerase in all examined NSCLC cell lines. LY3009120 also inhibited in vivo tumor growth in NSCLC cells harboring the BRAF non-V600E mutation. CONCLUSION: LY3009120 is a potent therapeutic agent for patients with BRAF non-V600E mutant NSCLC.

DOI： 10.21873/anticanres.14237

S100 Soil Sensor Receptors and Molecular Targeting Therapy Against Them in Cancer Metastasis. 査読国際誌

Nahoko Tomonobu, Rie Kinoshita, Masakiyo Sakaguchi

Translational oncology 13 ( 4 ) 100753 - 100753 2020年4月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The molecular mechanisms underlying the 'seed and soil' theory are unknown. S100A8/A9 (a heterodimer complex of S100A8 and S100A9 proteins that exhibits a 'soil signal') is a ligand for Toll-like receptor 4, causing distant melanoma cells to approach the lung as a 'seeding' site. Unknown soil sensors for S100A8/A9 may exist, e.g., extracellular matrix metalloproteinase inducer, neuroplastin, activated leukocyte cell adhesion molecule, and melanoma cell adhesion molecule. We call these receptor proteins 'novel S100 soil sensor receptors (novel SSSRs).' Here we review and summarize a crucial role of the S100A8/A9-novel SSSRs' axis in cancer metastasis. The binding of S100A8/A9 to individual SSSRs is important in cancer metastasis via upregulations of the epithelial-mesenchymal transition, cellular motility, and cancer cell invasiveness, plus the formation of an inflammatory immune suppressive environment in metastatic organ(s). These metastatic cellular events are caused by the SSSR-featured signal transductions we identified that provide cancer cells a driving force for metastasis. To deprive cancer cells of these metastatic forces, we developed novel biologics that prevent the interaction of S100A8/A9 with SSSRs, followed by the efficient suppression of S100A8/A9-mediated lung-tropic metastasis in vivo.

DOI： 10.1016/j.tranon.2020.100753

PLOD2 Is Essential to Functional Activation of Integrin β1 for Invasion/Metastasis in Head and Neck Squamous Cell Carcinomas. 査読国際誌

Yushi Ueki, Ken Saito, Hidekazu Iioka, Izumi Sakamoto, Yasuhiro Kanda, Masakiyo Sakaguchi, Arata Horii, Eisaku Kondo

iScience 23 ( 2 ) 100850 - 100850 2020年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Identifying the specific functional regulator of integrin family molecules in cancer cells is critical because they are directly involved in tumor invasion and metastasis. Here we report high expression of PLOD2 in oropharyngeal squamous cell carcinomas (SCCs) and its critical role as a stabilizer of integrin β1, enabling integrin β1 to initiate tumor invasion/metastasis. Integrin β1 stabilized by PLOD2-mediated hydroxylation was recruited to the plasma membrane, its functional site, and accelerated tumor cell motility, leading to tumor metastasis in vivo, whereas loss of PLOD2 expression abrogated it. In accordance with molecular analysis, examination of oropharyngeal SCC tissues from patients corroborated PLOD2 expression associated with integrin β1 at the invasive front of tumor nests. PLOD2 is thus implicated as the key regulator of integrin β1 that prominently regulates tumor invasion and metastasis, and it provides important clues engendering novel therapeutics for these intractable cancers.

DOI： 10.1016/j.isci.2020.100850

Caco-2 cells monolayer as an in-vitro model for probiotic strain translocation 査読国際共著国際誌

Ni Nengah Dwi Fatmawati, Kazuyoshi Goto, I. Putu Bayu Mayura, Komang Ayu Nocianitri, Yan Ramona, Masakiyo Sakaguchi, Osamu Matsushita, I. Nengah Sujaya

Bali Medical Journal 9 ( 1 ) 137 - 142 2020年

掲載種別：研究論文（学術雑誌）

Background: Caco-2 cells monolayer is one of in vitro models to evaluate the translocation capacity of Lactobacillus spp probiotic strains. The translocation is influenced by mucosa permeability of enterocytes as shown by increasing transepithelial resistance (TER) and formation of tight junction proteins. The pore size of the supported permeable membrane used in in vitro assay was one of the crucial factors in performing bacterial translocation assay. Almost no study has been conducted using Caco-2 cells monolayer grown on 8-μm pore size polycarbonate membrane for evaluating probiotics translocation. Therefore this study aimed to determine whether the Caco-2 cells monolayer model was suitable as an in vitro translocation model. Methods: Caco-2 cells monolayer was seeded onto 8-μm collagen-coated polycarbonate membrane insert Transwell®. Differentiation of Caco-2 cells was detected by measuring the TER, while the ZO-1 protein (the tight junction proteins) was detected by immunofluorescence. H2 O2 was used as a tight junction disruptive agent. Data were analyzed using SPSS version 23 software to compare the mean of TER measurement between untreated and H2 O2-treated Caco-2 cells monolayer. Results: The result showed that the TER of Caco-2 cells monolayer was gradually increasing until day 14, reaching more than 800 ohm.cm2. Furthermore, the ZO-1 protein was successfully detected, indicated the tight junction formation. TER value of H2 O2-treated cells showed significantly lower than that of untreated cells (P<0.05), indicating a disturbance of cells monolayer integrity. Lactobacillus rhamnosus FBB81 was used for validating the translocation. There was no translocation observed; however, translocation was observed in H2 O2-treated cells. Conclusion: Altogether suggests that Caco-2 cells grown on 8 μm-pore size permeable filters could be considered as a suitable in vitro model for probiotics strains translocation.

DOI： 10.15562/bmj.v9i1.1633

PODXL1 promotes metastasis of the pancreatic ductal adenocarcinoma by activating the C5aR/C5a axis from the tumor microenvironment. 査読国際誌

Ken Saito, Hidekazu Iioka, Satoshi Maruyama, I Wayan Sumardika, Masakiyo Sakaguchi, Eisaku Kondo

Neoplasia (New York, N.Y.) 21 ( 12 ) 1121 - 1132 2019年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

Pancreatic invasive ductal adenocarcinoma (PDAC) is a representative intractable malignancy under the current cancer therapies, and is considered a scirrhous carcinoma because it develops dense stroma. Both PODXL1, a member of CD34 family molecules, and C5aR, a critical cell motility inducer, have gained recent attention, as their expression was reported to correlate with poor prognosis for patients with diverse origins including PDAC; however, previous studies reported independently on their respective biological significance. Here we demonstrate that PODXL1 is essential for metastasis of PDAC cells through its specific interaction with C5aR. In vitro assay demonstrated that PODXL1 bound to C5aR, which stabilized C5aR protein and recruited it to cancer cell plasma membranes to receive C5a, an inflammatory chemoattractant factor. PODXL1 knockout in PDAC cells abrogated their metastatic property in vivo, emulating the liver metastatic mouse model treated with anti-C5a neutralizing antibody. In molecular studies, PODXL1 triggered EMT on PDAC cells in response to stimulation by C5a, corroborating PODXL1 involvement in PDAC cellular invasive properties via specific interaction with the C5aR/C5a axis. Confirming the molecular assays, histological examination showed coexpression of PODXL1 and C5aR at the invasive front of primary cancer nests as well as in liver metastatic foci of PDAC both in the mouse metastasis model and patient tissues. Hence, the novel direct interaction between PODXL1 and the C5aR/C5a axis may provide a better integrated understanding of PDAC biological characteristics including its tumor microenvironment factors.

DOI： 10.1016/j.neo.2019.09.003

Crumbs3 promotes metastasis of colon cancer by regulating focal adhesion components 査読

Iioka Hidekazu, Saito Ken, Sakaguchi Masakiyo, Morii Eiichi, Kondo Eisaku

CANCER SCIENCE 145 ( 10 ) 2740 - 2753 2019年11月

Crumbs3 is a critical factor that regulates invasion and metastasis of colon adenocarcinoma via the specific interaction with FGFR1. 査読国際誌

Hidekazu Iioka, Ken Saito, Masakiyo Sakaguchi, Taro Tachibana, Keiichi Homma, Eisaku Kondo

International journal of cancer 145 ( 10 ) 2740 - 2753 2019年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Epithelial cell polarity regulator Crumbs3 (Crb3), a mammalian homolog within the Drosophila Crb gene family, was initially identified as an essential embryonic development factor. It is recently implicated in tumor suppression, though its specific functions are controversial. We here demonstrate that Crb3 strongly promotes tumor invasion and metastasis of human colon adenocarcinoma cells. Crb3 centrality to tumor migration was supported by strong expression at invasive front and metastatic foci of colonic adenocarcinoma of the patient tissues. Accordingly, two different Crb3-knockout (KO) lines, Crb3-KO (Crb3 -/-) DLD-1 and Crb3-KO WiDr from human colonic adenocarcinomas, were generated by the CRISPR-Cas9 system. Crb3-KO DLD-1 cells exhibited loss of cellular mobility in vitro and dramatic suppression of liver metastases in vivo in contrast to the wild type of DLD-1. Unlike DLD-1, Crb3-KO WiDr mobility and metastasis were unaffected, which were similar to wild-type WiDr. Proteome analysis of Crb3-coimmunopreciptated proteins identified different respective fibroblast growth factor receptor (FGFR) isotypes specifically bound to Crb3 isoform a through their intracellular domain. In DLD-1, Crb3 showed membranous localization of FGFR1 leading to its functional activation, whereas Crb3 bound to cytoplasmic FGFR4 in WiDr without FGFR1 expression, leading to cellular growth. Correlative expression between Crb3 and FGFR1 was consistently detected in primary and metastatic colorectal cancer patient tissues. Taking these together, Crb3 critically accelerates cell migration, namely invasion and metastasis of human colon cancers, through specific interaction to FGFR1 on colon cancer cells.

DOI： 10.1002/ijc.32336

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年10月

Upregulation of Mobility in Pancreatic Cancer Cells by Secreted S100A11 Through Activation of Surrounding Fibroblasts. 査読国際共著国際誌

Yosuke Mitsui, Nahoko Tomonobu, Masami Watanabe, Rie Kinoshita, I Wayan Sumardika, Chen Youyi, Hitoshi Murata, Ken-Ichi Yamamoto, Takuya Sadahira, Acosta Gonzalez Herik Rodrigo, Hitoshi Takamatsu, Kota Araki, Akira Yamauchi, Masahiro Yamamura, Hideyo Fujiwara, Yusuke Inoue, Junichiro Futami, Ken Saito, Hidekazu Iioka, Eisaku Kondo, Masahiro Nishibori, Shinichi Toyooka, Yasuhiko Yamamoto, Yasutomo Nasu, Masakiyo Sakaguchi

Oncology research 27 ( 8 ) 945 - 956 2019年8月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

S100A11, a member of the S100 family of proteins, is actively secreted from pancreatic ductal adenocarcinoma (PDAC) cells. However, the role of the extracellular S100A11 in PDAC progression remains unclear. In the present study, we investigated the extracellular role of S100A11 in crosstalking between PDAC cells and surrounding fibroblasts in PDAC progression. An abundant S100A11 secreted from pancreatic cancer cells stimulated neighboring fibroblasts through receptor for advanced glycation end products (RAGE) upon S100A11 binding and was followed by not only an enhanced cancer cell motility in vitro but also an increased number of the PDAC-derived circulating tumor cells (CTCs) in vivo. Mechanistic investigation of RAGE downstream in fibroblasts revealed a novel contribution of a mitogen-activated protein kinase kinase kinase (MAPKKK), tumor progression locus 2 (TPL2), which is required for positive regulation of PDAC cell motility through induction of cyclooxygenase 2 (COX2) and its catalyzed production of prostaglandin E2 (PGE2), a strong chemoattractive fatty acid. The extracellularly released PGE2 from fibroblasts was required for the rise in cellular migration as well as infiltration of their adjacent PDAC cells in a coculture setting. Taken together, our data reveal a novel role of the secretory S100A11 in PDAC disseminative progression through activation of surrounding fibroblasts triggered by the S100A11-RAGE-TPL2-COX2 pathway. The findings of this study will contribute to the establishment of a novel therapeutic antidote to PDACs that are difficult to treat by regulating cancer-associated fibroblasts (CAFs) through targeting the identified pathway.

DOI： 10.3727/096504019X15555408784978

Newly developed anti-S100A8/A9 monoclonal antibody efficiently prevents lung tropic cancer metastasis. 査読国際共著国際誌

Rie Kinoshita, Hiroki Sato, Akira Yamauchi, Yuta Takahashi, Yusuke Inoue, I Wayan Sumardika, Youyi Chen, Nahoko Tomonobu, Kota Araki, Kazuhiko Shien, Shuta Tomida, Hidejiro Torigoe, Kei Namba, Eisuke Kurihara, Yusuke Ogoshi, Hitoshi Murata, Ken-Ichi Yamamoto, Junichiro Futami, Endy Widya Putranto, I Made Winarsa Ruma, Hiromasa Yamamoto, Junichi Soh, Toshihiko Hibino, Masahiro Nishibori, Eisaku Kondo, Shinichi Toyooka, Masakiyo Sakaguchi

International journal of cancer 145 ( 2 ) 569 - 575 2019年7月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The metastatic dissemination of cancer cells to remote areas of the body is the most problematic aspect in cancer patients. Among cancers, melanomas are notoriously difficult to treat due to their significantly high metastatic potential even during early stages. Hence, the establishment of advanced therapeutic approaches to regulate metastasis is required to overcome the melanoma disease. An accumulating mass of evidence has indicated a critical role of extracellular S100A8/A9 in melanoma distant metastasis. Lung S100A8/A9 is induced by melanoma cells from distant organs and it attracts these cells to its enriched lung environment since melanoma cells possess several receptors that sense the S100A8/A9 ligand. We hence aimed to develop a neutralizing antibody against S100A8/A9 that would efficiently block melanoma lung metastasis. Our protocol provided us with one prominent antibody, Ab45 that efficiently suppressed not only S100A8/A9-mediated melanoma mobility but also lung tropic melanoma metastasis in a mouse model. This prompted us to make chimeric Ab45, a chimera antibody consisting of mouse Ab45-Fab and human IgG2-Fc. Chimeric Ab45 also showed significant inhibition of the lung metastasis of melanoma. From these results, we have high hopes that the newly produced antibody will become a potential biological tool to block melanoma metastasis in future clinical settings.

DOI： 10.1002/ijc.31982

Convenient methodology for extraction and subsequent selective propagation of mouse melanocytes in culture from adult mouse skin tissue. 査読国際共著国際誌

Nahoko Tomonobu, Rie Kinoshita, I Wayan Sumardika, Youyi Chen, Yusuke Inoue, Akira Yamauchi, Ken-Ichi Yamamoto, Hitoshi Murata, Masakiyo Sakaguchi

Biochemistry and biophysics reports 18 100619 - 100619 2019年7月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Mouse melanoma B16-BL6 cells are useful cells for cancer metastatic studies. To understand the metastatic principle at molecular levels, it is necessary to carry out experiments in which cancer cells and their normal counterparts are compared. However, unlike normal human melanocytes, preparation of normal mouse melanocytes is quite difficult due to the lack of marketing and insufficient information on an established protocol for primary culture of mouse melanocytes. In this study, we aimed to establish a convenient method for primary culture of mouse melanocytes on the basis of the protocol for human melanocytes. The main obstacles to preparing pure mouse melanocytes are how to digest mouse skin tissue and how to reduce the contamination of keratinocytes and fibroblasts. The obstacles were overcome by collagenase digestion for skin specimens, short time trypsinization for separating melanocytes and keratinocytes, and use of 12-O-Tetradecanoylphorbol 13-acetate (TPA) and cholera toxin in the culture medium. These supplements act to prevent the proliferation of keratinocytes and fibroblasts, respectively. The convenient procedure enabled us to prepare a pure culture of normal mouse melanocytes. Using enriched normal mouse melanocytes and cancerous B16-BL6 cells, we compared the expression levels of melanoma cell adhesion molecule (MCAM), an important membrane protein for melanoma metastasis, in the cells. The results showed markedly higher expression of MCAM in B16-BL6 cells than in normal mouse melanocytes.

DOI： 10.1016/j.bbrep.2019.100619

Critical role of the MCAM-ETV4 axis triggered by extracellular S100A8/A9 in breast cancer aggressiveness. 査読国際共著国際誌

Youyi Chen, I Wayan Sumardika, Nahoko Tomonobu, Rie Kinoshita, Yusuke Inoue, Hidekazu Iioka, Yosuke Mitsui, Ken Saito, I Made Winarsa Ruma, Hiroki Sato, Akira Yamauchi, Hitoshi Murata, Ken-Ichi Yamamoto, Shuta Tomida, Kazuhiko Shien, Hiromasa Yamamoto, Junichi Soh, Junichiro Futami, Miyoko Kubo, Endy Widya Putranto, Takashi Murakami, Ming Liu, Toshihiko Hibino, Masahiro Nishibori, Eisaku Kondo, Shinichi Toyooka, Masakiyo Sakaguchi

Neoplasia (New York, N.Y.) 21 ( 7 ) 627 - 640 2019年7月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Metastatic breast cancer is the leading cause of cancer-associated death in women. The progression of this fatal disease is associated with inflammatory responses that promote cancer cell growth and dissemination, eventually leading to a reduction of overall survival. However, the mechanism(s) of the inflammation-boosted cancer progression remains unclear. In this study, we found for the first time that an extracellular cytokine, S100A8/A9, accelerates breast cancer growth and metastasis upon binding to a cell surface receptor, melanoma cell adhesion molecule (MCAM). Our molecular analyses revealed an important role of ETS translocation variant 4 (ETV4), which is significantly activated in the region downstream of MCAM upon S100A8/A9 stimulation, in breast cancer progression in vitro as well as in vivo. The MCAM-mediated activation of ETV4 induced a mobile phenotype called epithelial-mesenchymal transition (EMT) in cells, since we found that ETV4 transcriptionally upregulates ZEB1, a strong EMT inducer, at a very high level. In contrast, downregulation of either MCAM or ETV4 repressed EMT, resulting in greatly weakened tumor growth and lung metastasis. Overall, our results revealed that ETV4 is a novel transcription factor regulated by the S100A8/A9-MCAM axis, which leads to EMT through ZEB1 and thereby to metastasis in breast cancer cells. Thus, therapeutic strategies based on our findings might improve patient outcomes.

DOI： 10.1016/j.neo.2019.04.006

Melanoma cell adhesion molecule is the driving force behind the dissemination of melanoma upon S100A8/A9 binding in the original skin lesion. 査読国際共著国際誌

Youyi Chen, I Wayan Sumardika, Nahoko Tomonobu, I Made Winarsa Ruma, Rie Kinoshita, Eisaku Kondo, Yusuke Inoue, Hiroki Sato, Akira Yamauchi, Hitoshi Murata, Ken-Ichi Yamamoto, Shuta Tomida, Kazuhiko Shien, Hiromasa Yamamoto, Junichi Soh, Ming Liu, Junichiro Futami, Kaori Sasai, Hiroshi Katayama, Miyoko Kubo, Endy Widya Putranto, Toshihiko Hibino, Bei Sun, Masahiro Nishibori, Shinichi Toyooka, Masakiyo Sakaguchi

Cancer letters 452 178 - 190 2019年6月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Since metastasis accounts for the majority of cancer-associated deaths, studies on the mechanisms of metastasis are needed to establish innovative strategies for cancer treatment. We previously reported that melanoma cell adhesion molecule (MCAM) functions as a critical receptor for S100A8/A9, and binding of S100A8/A9 to MCAM results in the migration of melanoma cells to lung tissue. However, the critical role of MCAM in the original melanoma skin lesion is still not clear. In this study, we aimed to determine the importance of the S100A8/A9-MCAM axis in melanoma dissemination in a skin lesion as a critical early step for metastasis. Mechanistic studies revealed the downstream signaling of MCAM that signaled the induction of metastasis. S100A8/A9-MCAM binding activates mitogen-activated protein kinase kinase kinase 8 (MAP3K8), also termed TPL2, leading to strong activation of the transcription factor ETV4 and subsequent induction of matrix metalloproteinase-25 (MMP25), and finally to induction of melanoma lung tropic metastasis. Collectively, our results demonstrate a crucial role of the S100A8/A9-MCAM signaling axis in metastatic onset of melanoma cells and indicate that strategies targeting the identified pathway may be useful for the establishment of innovative anti-cancer therapies.

DOI： 10.1016/j.canlet.2019.03.023

Extracellular S100A11 Plays a Critical Role in Spread of the Fibroblast Population in Pancreatic Cancers. 査読国際共著国際誌

Hitoshi Takamatsu, Ken-Ichi Yamamoto, Nahoko Tomonobu, Hitoshi Murata, Yusuke Inoue, Akira Yamauchi, I Wayan Sumardika, Youyi Chen, Rie Kinoshita, Masahiro Yamamura, Hideyo Fujiwara, Yosuke Mitsui, Kota Araki, Junichiro Futami, Ken Saito, Hidekazu Iioka, I Made Winarsa Ruma, Endy Widya Putranto, Masahiro Nishibori, Eisaku Kondo, Yasuhiko Yamamoto, Shinichi Toyooka, Masakiyo Sakaguchi

Oncology research 27 ( 6 ) 713 - 727 2019年6月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The fertile stroma in pancreatic ductal adenocarcinomas (PDACs) has been suspected to greatly contribute to PDAC progression. Since the main cell constituents of the stroma are fibroblasts, there is crosstalking(s) between PDAC cells and surrounding fibroblasts in the stroma, which induces a fibroblast proliferation burst. We have reported that several malignant cancer cells including PDAC cells secrete a pronounced level of S100A11, which in turn stimulates proliferation of cancer cells via the receptor for advanced glycation end products (RAGE) in an autocrine manner. Owing to the RAGE+ expression in fibroblasts, the extracellular abundant S100A11 will affect adjacent fibroblasts. In this study, we investigated the significance of the paracrine axis of S100A11-RAGE in fibroblasts for their proliferation activity. In in vitro settings, extracellular S100A11 induced upregulation of fibroblast proliferation. Our mechanistic studies revealed that the induction is through RAGE-MyD88-mTOR-p70 S6 kinase upon S100A11 stimulation. The paracrine effect on fibroblasts is linked mainly to triggering growth but not cellular motility. Thus, the identified pathway might become a potential therapeutic target to suppress PDAC progression through preventing PDAC-associated fibroblast proliferation.

DOI： 10.3727/096504018X15433161908259

exSSSRs (extracellular S100 soil sensor receptors)-Fc fusion proteins work as prominent decoys to S100A8/A9-induced lung tropic cancer metastasis. 査読国際共著国際誌

Rie Kinoshita, Hiroki Sato, Akira Yamauchi, Yuta Takahashi, Yusuke Inoue, I Wayan Sumardika, Youyi Chen, Nahoko Tomonobu, Kota Araki, Kazuhiko Shien, Shuta Tomida, Hidejiro Torigoe, Kei Namba, Eisuke Kurihara, Yusuke Ogoshi, Hitoshi Murata, Ken-Ichi Yamamoto, Junichiro Futami, Endy Widya Putranto, I Made Winarsa Ruma, Hiromasa Yamamoto, Junichi Soh, Toshihiko Hibino, Masahiro Nishibori, Eisaku Kondo, Shinichi Toyooka, Masakiyo Sakaguchi

International journal of cancer 144 ( 12 ) 3138 - 3145 2019年6月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Within the "seed and soil" theory of organ tropic cancer metastasis is a growing compilation of evidence that S100A8/A9 functions as a soil signal that attracts cancer cells to certain organs, which prove beneficial to their growth. S100A8/A9-sensing receptors including Toll-like receptor 4 (TLR4), advanced glycation end products (RAGE), and also important receptors we recently succeeded in identifying (EMMPRIN, NPTNβ, MCAM, and ALCAM) have the potential to become promising therapeutic targets. In our study, we prepared extracellular regions of these novel molecules and fused them to human IgG2-Fc to extend half-life expectancy, and we evaluated the anti-metastatic effects of the purified decoy proteins on metastatic cancer cells. The purified proteins markedly suppressed S100A8/A9-mediated lung tropic cancer metastasis. We hence expect that our novel biologics may become a prominent medicine to prevent cancer metastasis in clinical settings through cutting the linkage between "seed and soil".

DOI： 10.1002/ijc.31945

Neuroplastin-β mediates S100A8/A9-induced lung cancer disseminative progression. 査読国際共著国際誌

Sumardika IW, Chen Y, Tomonobu N, Kinoshita R, Ruma IMW, Sato H, Kondo E, Inoue Y, Yamauchi A, Murata H, Yamamoto KI, Tomida S, Shien K, Yamamoto H, Soh J, Futami J, Putranto EW, Hibino T, Nishibori M, Toyooka S, Sakaguchi M

Mol Carcinog. 58 ( 6 ) 980 - 995 2019年6月

Histidine-rich glycoprotein augments natural killer cell function by modulating PD-1 expression via CLEC-1B. 査読国際誌

Nishimura Y, Wake H, Teshigawara K, Wang D, Sakaguchi M, Otsuka F, Nishibori M

Pharmacol Res Perspect. 7 ( 3 ) e00481 2019年5月

Hepatocyte nuclear factor 4α regulates megalin expression in proximal tubular cells. 査読国際誌

Sasaki S, Hara A, Sakaguchi M, Nangaku M, Inoue Y

Biochem Biophys Rep. 17 87 - 92 2019年3月

Anti-tumor effect of neratinib against lung cancer cells harboring HER2 oncogene alterations. 査読国際誌

Yusuke Ogoshi, Kazuhiko Shien, Takahiro Yoshioka, Hidejiro Torigoe, Hiroki Sato, Masakiyo Sakaguchi, Shuta Tomida, Kei Namba, Eisuke Kurihara, Yuta Takahashi, Ken Suzawa, Hiromasa Yamamoto, Junichi Soh, Shinichi Toyooka

Oncology letters 17 ( 3 ) 2729 - 2736 2019年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

Human epidermal growth factor receptor 2 (HER2) is a member of the ErbB family of receptor tyrosine kinases. Numerous studies have reported the amplification and overexpression of HER2 in several types of cancer, including non-small cell lung cancer (NSCLC). However, the benefits of HER2-targeted therapy have not been fully established. In the present study, the anti-tumor effect of neratinib, an irreversible pan-HER tyrosine kinase inhibitor (TKI), against NSCLC cells harboring HER2 alterations was investigated. The sensitivity of normal bronchial epithelial cells (BEAS-2B) ectopically overexpressing wild-type or mutant HER2 to neratinib was assessed. Furthermore, the anti-tumor activity of neratinib in several NSCLC cell lines harboring HER2 alterations was determined in vitro and in vivo, and the association between their genetic alterations and sensitivity to neratinib treatment was investigated. BEAS-2B cells ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, G660D and S310F) exhibited constitutive autophosphorylation of HER2, as determined by western blotting. While these BEAS-2B cells were sensitive to neratinib, they were insensitive to erlotinib, a first-generation epidermal growth factor receptor-TKI. Neratinib also exerted anti-proliferative effects on HER2-altered (H2170, Calu-3 and H1781) NSCLC cell lines. Neratinib was also demonstrated to exert strong tumor growth inhibitory activity in mouse xenograft models using HER2-altered lung cancer cells. The results of the present study strongly suggest that neratinib has potential as a promising therapeutic option for the treatment of HER2-altered NSCLC.

DOI： 10.3892/ol.2019.9908

JNK-mediated phosphorylation of SARM1 regulates NAD(+) cleavage activity to inhibit mitochondrial respiration. 査読国際誌

Murata H, Khine CC, Nishikawa A, Yamamoto KI, Kinoshita R, Sakaguchi M

J Biol Chem 293 ( 49 ) 18933 - 18943 2018年12月

Combined inhibition of MEK and PI3K pathways overcomes acquired resistance to EGFR-TKIs in non-small cell lung cancer. 査読国際誌

Hiroki Sato, Hiromasa Yamamoto, Masakiyo Sakaguchi, Kazuhiko Shien, Shuta Tomida, Tadahiko Shien, Hirokuni Ikeda, Minami Hatono, Hidejiro Torigoe, Kei Namba, Takahiro Yoshioka, Eisuke Kurihara, Yusuke Ogoshi, Yuta Takahashi, Junichi Soh, Shinichi Toyooka

Cancer science 109 ( 10 ) 3183 - 3196 2018年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Compensatory activation of the signal transduction pathways is one of the major obstacles for the targeted therapy of non-small cell lung cancer (NSCLC). Herein, we present the therapeutic strategy of combined targeted therapy against the MEK and phosphoinositide-3 kinase (PI3K) pathways for acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in NSCLC. We investigated the efficacy of combined trametinib plus taselisib therapy using experimentally established EGFR-TKI-resistant NSCLC cell lines. The results showed that the feedback loop between MEK/ERK and PI3K/AKT pathways had developed in several resistant cell lines, which caused the resistance to single-agent treatment with either inhibitor alone. Meanwhile, the combined therapy successfully regulated the compensatory activation of the key intracellular signals and synergistically inhibited the cell growth of those cells in vitro and in vivo. The resistance mechanisms for which the dual kinase inhibitor therapy proved effective included (MET) mesenchymal-epithelial transition factor amplification, induction of epithelial-to-mesenchymal transition (EMT) and EGFR T790M mutation. In further analysis, the combination therapy induced the phosphorylation of p38 MAPK signaling, leading to the activation of apoptosis cascade. Additionally, long-term treatment with the combination therapy induced the conversion from EMT to mesenchymal-to-epithelial transition in the resistant cell line harboring EMT features, restoring the sensitivity to EGFR-TKI. In conclusion, our results indicate that the combined therapy using MEK and PI3K inhibitors is a potent therapeutic strategy for NSCLC with the acquired resistance to EGFR-TKIs.

DOI： 10.1111/cas.13763

Induction of hepatic metabolic functions by a novel variant of hepatocyte nuclear factor 4γ. 査読

Sasaki S, Urabe M, Maeda T, Suzuki J, Irie R, Suzuki M, Tomaru Y, Sakaguchi M, Gonzalez FJ, Inoue Y

Molecular and cellular biology 38 ( 24 ) e00213 - e00218 2018年9月

EGFR変異陽性肺癌におけるLRIG1の抗腫瘍効果(Tumor-suppressive effect of LRIG1 in non-small cell lung cancer harboring mutant EGFR)

鳥越英次郎, 山本寛斉, 阪口政清, 難波圭, 佐藤博紀, 枝園和彦, 諏澤憲, 宗淳一, 冨田秀太, 佃和憲, 三好新一郎, 豊岡伸一

日本癌学会総会記事 77回 940 - 940 2018年9月

Embigin Promotes Prostate Cancer Progression by S100A4-Dependent and-Independent Mechanisms. 査読国際誌

I Made Winarsa Ruma, Rie Kinoshita, Nahoko Tomonobu, Yusuke Inoue, Eisaku Kondo, Akira Yamauchi, Hiroki Sato, I Wayan Sumardika, Youyi Chen, Ken-Ichi Yamamoto, Hitoshi Murata, Shinichi Toyooka, Masahiro Nishibori, Masakiyo Sakaguchi

Cancers 10 ( 7 ) 2018年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

Embigin, a transmembrane glycoprotein belonging to the immunoglobulin superfamily, is involved in prostate and mammary gland development. As embigin's roles in cancer remain elusive, we studied its biological functions and interaction with extracellular S100A4 in prostate cancer progression. We found by a pull-down assay that embigin is a novel receptor for S100A4, which is one of the vital cancer microenvironment milleu. Binding of extracellular S100A4 to embigin mediates prostate cancer progression by inhibition of AMPK activity, activation of NF-κB, MMP9 and mTORC1 signaling, and inhibition of autophagy, which increase prostate cancer cell motility. We also found that embigin promotes prostate cancer growth, spheroid- and colony-forming ability, and survival upon chemotherapy independently of S100A4. An in vivo growth mouse model confirmed the importance of embigin and its cytoplasmic tail in mediating prostate tumor growth. Moreover, embigin and p21WAF1 can be used to predict survival of prostate cancer patients. Our results demonstrated for the first time that the S100A4-embigin/AMPK/mTORC1/p21WAF1 and NF-κB/MMP9 axis is a vital oncogenic molecular cascade for prostate cancer progression. We proposed that embigin and p21WAF1 could be used as prognostic biomarkers and a strategy to inhibit S100A4-embigin binding could be a therapeutic approach for prostate cancer patients.

DOI： 10.3390/cancers10070239

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年7月

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年7月

Tumor-suppressive effect of LRIG1, a negative regulator of ErbB, in non-small cell lung cancer harboring mutant EGFR. 査読国際誌

Hidejiro Torigoe, Hiromasa Yamamoto, Masakiyo Sakaguchi, Chen Youyi, Kei Namba, Hiroki Sato, Kazuhiko Shien, Junichi Soh, Ken Suzawa, Shuta Tomida, Kazunori Tsukuda, Shinichiro Miyoshi, Shinichi Toyooka

Carcinogenesis 39 ( 5 ) 719 - 727 2018年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Epidermal growth factor receptor (EGFR) is a member of the ErbB (HER) family that is known to play important roles in the pathogenesis of various human cancers. Mutations of the EGFR gene are commonly found as oncogenic driver mutations and have been targeted for treatment of non-small cell lung cancer (NSCLC). Leucine-rich repeat and immunoglobulin-like domain protein-1 (LRIG1) is a cell-surface protein that is known as a negative regulator of the ErbB (HER) family. In this study, we first confirmed that the expression levels of LRIG1 were much lower in NSCLC than in non-malignant cells or tissues. Next, we focused on the effect of LRIG1 in NSCLC. For this purpose, we established clones stably overexpressing LRIG1, using EGFR-mutant (HCC827, HCC4011 and NCI-H1975) and wild-type (A549) cells. Transfection of LRIG1 was associated with a decrease in the expression and phosphorylation levels of EGFR in the HCC827, HCC4011 and NCI-H1975 cells. It was also associated with strong suppression of the cell proliferative, invasive, migratory and tumorigenic potential of the HCC827 cells. On the other hand, no such effects were observed in the A549 cells. In addition, LRIG1 also downregulated the expression and phosphorylation levels of other tyrosine kinase receptors, such as HER2, HER3, MET and IGF-1R, and prevented the epithelial-to-mesenchymal transition induced by TGF-β in the HCC827 cells. These findings suggest that LRIG1 exerts important tumor-suppressive effects in EGFR-mutant NSCLC and has the potential to become a novel therapeutic target for EGFR-mutant NSCLC.

DOI： 10.1093/carcin/bgy044

Tumor necrosis factor-α downregulates the REIC/Dkk-3 tumor suppressor gene in normal human skin keratinocytes. 査読

Kataoka K, Maehara N, Ayabe Y, Murata H, Huh NH, Sakaguchi M

Molecular medicine reports 17 ( 5 ) 6661 - 6666 2018年5月

Therapeutic strategies for afatinib-resistant lung cancer harboring HER2 alterations 査読

Hidejiro Torigoe, Kazuhiko Shien, Tatsuaki Takeda, Takahiro Yoshioka, Kei Namba, Hiroki Sato, Ken Suzawa, Hiromasa Yamamoto, Junichi Soh, Masakiyo Sakaguchi, Shuta Tomida, Kazunori Tsukuda, Shinichiro Miyoshi, Shinichi Toyooka

Cancer Science 109 ( 5 ) 1493 - 1502 2018年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Blackwell Publishing Ltd

Human epidermal growth factor receptor 2 (HER2) plays an important role in the pathogenesis of various cancers. HER2 alterations have been suggested to be a therapeutic target in non-small-cell lung cancer (NSCLC), just as in breast and gastric cancers. We previously reported that the pan-HER inhibitor afatinib could be a useful therapeutic agent as HER2-targeted therapy for patients with NSCLC harboring HER2 alterations. However, acquired resistance to afatinib was observed in the clinical setting, similar to the case for other HER inhibitors. Thus, elucidation of the mechanisms underlying the development of acquired drug resistance and exploring means to overcome acquired drug resistance are important issues in the treatment of NSCLC. In this study, we experimentally established afatinib-resistant cell lines from NSCLC cell lines harboring HER2 alterations, and investigated the mechanisms underlying the acquisition of drug resistance. The established cell lines showed several unique afatinib-resistance mechanisms, including MET amplification, loss of HER2 amplification and gene expression, epithelial-to-mesenchymal transition (EMT) and acquisition of cancer stem cell (CSC)-like features. The afatinib-resistant cell lines showing MET amplification were sensitive to the combination of afatinib plus crizotinib (a MET inhibitor), both in vitro and in vivo. The resistant cell lines which showed EMT or had acquired CSC-like features remained sensitive to docetaxel, like the parental cells. These findings may provide clues to countering the resistance to afatinib in NSCLC patients with HER2 alterations.

DOI： 10.1111/cas.13571

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

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

Oncology research 26 ( 3 ) 431 - 444 2018年4月

肺腺がんにおけるSpred2の役割の解明

太田陽子, 藤澤真義, 吉村禎造, スマルディカ・イワヤン, 枝園和彦, 阪口政清, 豊岡伸一, 松川昭博

日本病理学会会誌 107 ( 1 ) 414 - 414 2018年4月

Effects of Histidine-rich glycoprotein on erythrocyte aggregation and hemolysis: Implications for a role under septic conditions 査読

Hui Zhong, Hidenori Wake, Keyue Liu, Yuan Gao, Kiyoshi Teshigawara, Masakiyo Sakaguchi, Shuji Mori, Masahiro Nishibori

Journal of Pharmacological Sciences 136 ( 3 ) 97 - 106 2018年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Japanese Pharmacological Society

The apoptotic process of erythrocytes is known as eryptosis, and is characterized by phosphatidylserine (PS) expression on the outer membrane. PS-positive erythrocytes are increased in sepsis, and PS is believed to facilitate coagulation of erythrocytes and activate macrophages. However, the relationship between eryptosis and abnormal coagulation in sepsis is still not fully understood. Histidine-rich glycoprotein (HRG) inhibits immunothrombus formation by regulating neutrophils and vascular endothelial cells. In the present study, we subjected isolated erythrocytes to Zn2+ stimulation, which activated their aggregation and PS expression. We then determined the Zn2+ contents in septic lung and kidney tissues, and found that they were elevated, suggesting that eryptosis was enhanced in these tissues. Erythrocyte adhesion to endothelial cells was also significantly increased after Zn2+ stimulation, and this effect was inhibited by HRG. Finally, we examined HRG treatment in septic model mice, and found that HRG decreased hemolysis, possibly due to its ability to bind heme. Our study demonstrated a novel Zn2+-initiated aggregation/thrombus formation pathway. We also showed the regulatory role of HRG in this pathway, together with the ability of HRG to inhibit hemolysis under septic conditions. HRG supplementation might be a novel therapeutic strategy for inflammatory disorders, especially sepsis.

DOI： 10.1016/j.jphs.2017.11.003

Therapeutic potential of targeting S100A11 in malignant pleural mesothelioma 査読

Hiroki Sato, Masakiyo Sakaguchi, Hiromasa Yamamoto, Shuta Tomida, Keisuke Aoe, Kazuhiko Shien, Takahiro Yoshioka, Kei Namba, Hidejiro Torigoe, Junichi Soh, Kazunori Tsukuda, Hiroyuki Tao, Kazunori Okabe, Shinichiro Miyoshi, Harvey I. Pass, Shinichi Toyooka

Oncogenesis 7 ( 1 ) 11 2018年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Publishing Group

Malignant pleural mesothelioma (MPM) is an aggressive tumor with an unfavorable prognosis. The standard therapeutic approaches are limited to surgery, chemotherapy, and radiotherapy. Because the consequent clinical outcome is often unsatisfactory, a different approach in MPM treatment is required. S100A11, a Ca2+-binding small protein with two EF-hands, is frequently upregulated in various human cancers. Interestingly, it has been found that intracellular and extracellular S100A11 have different functions in cell viability. In this study, we focused on the impact of extracellular S100A11 in MPM and explored the therapeutic potential of an S100A11-targeting strategy. We examined the secretion level of S100A11 in various kinds of cell lines by enzyme-linked immunosorbent assay. Among them, six out of seven MPM cell lines actively secreted S100A11, whereas normal mesothelial cell lines did not secrete it. To investigate the role of secreted S100A11 in MPM, we inhibited its function by neutralizing S100A11 with an anti-S100A11 antibody. Interestingly, the antibody significantly inhibited the proliferation of S100A11-secreting MPM cells in vitro and in vivo. Microarray analysis revealed that several pathways including genes involved in cell proliferation were negatively enriched in the antibody-treated cell lines. In addition, we examined the secretion level of S100A11 in various types of pleural effusions. We found that the secretion of S100A11 was significantly higher in MPM pleural effusions, compared to others, suggesting the possibility for the use of S100A11 as a biomarker. In conclusion, our results indicate that extracellular S100A11 plays important roles in MPM and may be a therapeutic target in S100A11-secreting MPM.

DOI： 10.1038/s41389-017-0017-3

Development of novel biologics for cancer metastasis via prevention of extracellular S100A8/A9 function 査読

Kinoshita Rie, Yamauchi Akira, Shien Kazuhiko, Tomida Shuta, Toyooka Shinichi, Kondo Eisaku, Sakaguchi Masakiyo

CANCER SCIENCE 109 1017 2018年1月

Crumbs3 promotes metastasis of colon cancer by regulating focal adhesion components 査読

Iioka Hidekazu, Saito Ken, Sakaguchi Masakiyo, Morii Eiichi, Kondo Eisaku

CANCER SCIENCE 109 1039 2018年1月

Stromal mesenchymal stem cells facilitate pancreatic cancer progression by regulating specific secretory molecules through mutual cellular interaction. 査読国際誌

Ken Saito, Masakiyo Sakaguchi, Satoshi Maruyama, Hidekazu Iioka, Endy Widya Putranto, I Wayan Sumardika, Nahoko Tomonobu, Takashi Kawasaki, Keiichi Homma, Eisaku Kondo

Journal of Cancer 9 ( 16 ) 2916 - 2929 2018年

記述言語：英語

Pancreatic ductal adenocarcinoma (PDAC) is currently one of the most intractable malignancies with a typical scirrhous pattern in histology. Due to its abundant tumor stroma and scant vascularization, chemotherapeutic agents are considered inefficiently permeable to cancer nests, making it highly difficult to cure the patients with PDAC. However, PDAC is also considered to owe its intractability to other critical factors such as cellular interaction between tumor cells and tumor microenvironment as well as architectural barriers, which increases in therapeutic resistance. Here, we report a specific cellular interaction between PDAC cells and mesenchymal stem cells (MSCs) intermingled in PDAC stroma, which facilitates cancer invasion. Secretory phenotype profiling revealed that production of Amphiregulin (AREG) and MMP-3 were specifically upregulated under the coexistence of BxPC3 cells with human MSCs (approximately four to ten folds in AREG, and twenty to sixty-folds in MMP-3 compared to that of BxPC3 cells alone), whereas MMP-9 expression was decreased (less than one-tenth comparing with that of BxPC3 cells alone). Blockage of AREG production by its specific siRNA removed MSC-mediated driving force of BxPC3 invasiveness. Immunohistochemical analysis of tissue samples obtained both from PDAC patients and PDAC imitating mouse xenografted models revealed that significant coexpression of AREG and its receptor EGFR were detected on the cancer cells at invasive front. These results strongly suggested that cellular interaction between cancer cells and MSCs in the PDAC stroma might be critical to cancer progression, especially in the process of local invasion and the early stage development of metastasis.

DOI： 10.7150/jca.24415

Crucial role of RAGE in inappropriate increase of smooth muscle cells from patients with pulmonary arterial hypertension. 査読国際誌

Kazufumi Nakamura, Masakiyo Sakaguchi, Hiromi Matsubara, Satoshi Akagi, Toshihiro Sarashina, Kentaro Ejiri, Kaoru Akazawa, Megumi Kondo, Koji Nakagawa, Masashi Yoshida, Toru Miyoshi, Takeshi Ogo, Takahiro Oto, Shinichi Toyooka, Yuichiro Higashimoto, Kei Fukami, Hiroshi Ito

PloS one 13 ( 9 ) e0203046 2018年

記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: Pulmonary vascular remodeling of pulmonary arterial hypertension (PAH) is characterized by an inappropriate increase of vascular cells. The receptor for advanced glycation end products (RAGE) is a type I single-pass transmembrane protein belonging to the immunoglobulin superfamily and is involved in a broad range of hyperproliferative diseases. RAGE is also implicated in the etiology of PAH and is overexpressed in pulmonary artery smooth muscle cells (PASMCs) in patients with PAH. We examined the role of RAGE in the inappropriate increase of PASMCs in patients with PAH. METHODS AND RESULTS: PASMCs were obtained from 12 patients with PAH including 9 patients with idiopathic PAH (IPAH) and 3 patients with heritable PAH (HPAH) (2 patients with BMPR2 mutation and one patient with SMAD9 mutation) who underwent lung transplantation. Western blot analysis and immunofluorescence staining revealed that RAGE and S100A8 and A9, ligands of RAGE, were overexpressed in IPAH and HPAH-PASMCs in the absence of any external growth stimulus. PDGF-BB (10 ng/mL) up-regulated the expression of RAGE in IPAH and HPAH-PASMCs. PAH-PASMCs are hyperplastic in the absence of any external growth stimulus as assessed by 3H-thymidine incorporation. This result indicates overgrowth characterized by continued growth under a condition of no growth stimulation in PAH-PASMCs. PDGF-BB stimulation caused a higher growth rate of PAH-PASMCs than that of non-PAH-PASMCs. AS-1, an inhibitor of TIR domain-mediated RAGE signaling, significantly inhibited overgrowth characterized by continued growth under a condition of no growth stimulation in IPAH and HPAH-PASMCs (P<0.0001). Furthermore, AS-1 significantly inhibited PDGF-stimulated proliferation of IPAH and HPAH-PASMCs (P<0.0001). CONCLUSIONS: RAGE plays a crucial role in the inappropriate increase of PAH-PASMCs. Inhibition of RAGE signaling may be a new therapeutic strategy for PAH.

DOI： 10.1371/journal.pone.0203046

β-1,3-Galactosyl-

Sumardika, I. W., Youyi, C., Kondo, E., Inoue, Y., Ruma, I. M. W., Murata, H., Kinoshita, R., Yamamoto, K. I., Tomida, S., Shien, K., Sato, H., Yamauchi, A., Futami, J., Putranto, E. W., Hibino, T., Toyooka, S., Nishibori, M., Sakaguchi, M.

Oncol Res 26 ( 3 ) 2018年

Signal Diversity of Receptor for Advanced Glycation End Products 国際誌

Masakiyo Sakaguchi, Rie Kinoshita, Endy Widya Putranto, I. Made Winarsa Ruma, I. Wayan Sumardika, Chen Youyi, Naoko Tomonobu, Ken-ichi Yamamoto, Hitoshi Murata

ACTA MEDICA OKAYAMA 71 ( 6 ) 459 - 465 2017年12月

担当区分：筆頭著者記述言語：英語出版者・発行元：OKAYAMA UNIV MED SCHOOL

The receptor for advanced glycation end products (RAGE) is involved in inflammatory pathogenesis. It functions as a receptor to multiple ligands such as AGEs, HMGB1 and S100 proteins, activating multiple intracellular signaling pathways with each ligand binding. The molecular events by which ligand-activated RAGE controls diverse signaling are not well understood, but some progress was made recently. Accumulating evidence revealed that RAGE has multiple binding partners within the cytoplasm and on the plasma membrane. It was first pointed out in 2008 that RAGE's cytoplasmic tail is able to recruit Diaphanous-1 (Dia-1), resulting in the acquisition of increased cellular motility through Rac1/Cdc42 activation. We also observed that within the cytosol, RAGE's cytoplasmic tail behaves similarly to a Toll-like receptor (TLR4)-TIR domain, interacting with TIRAP and MyD88 adaptor molecules that in turn activate multiple downstream signals. Subsequent studies demonstrated the presence of an alternative adaptor molecule, DAP10, on the plasma membrane. The coupling of RAGE with DAP10 is critical for enhancing the RAGE-mediated survival signal. Interestingly, RAGE interaction on the membrane was not restricted to DAP10 alone. The chemotactic G-protein-coupled receptors (GPCRs) formyl peptide receptors1 and 2 (FPR1 and FPR2) also interacted with RAGE on the plasma membrane. Binding interaction between leukotriene B4 receptor 1 (BLT1) and RAGE was also demonstrated. All of the interactions affected the RAGE signal polarity. These findings indicate that functional interactions between RAGE and various molecules within the cytoplasmic area or on the membrane area coordinately regulate multiple ligand-mediated RAGE responses, leading to typical cellular phenotypes in several pathological settings. Here we review RAGE's signaling diversity, to contribute to the understanding of the elaborate functions of RAGE in physiological and pathological contexts.

DOI： 10.18926/AMO/55582

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

Xu J, Sadahira T, Kinoshita R, Li SA, Huang P, Wada K, Araki M, Ochiai K, Noguchi H, Sakaguchi M, Nasu Y, Watanabe M

Oncology letters 14 ( 5 ) 5638 - 5642 2017年11月

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

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年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY

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

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

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年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

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

Robust cancer-specific gene expression by a novel cassette with hTERT and CMV promoter elements 査読

Masakiyo Sakaguchi, Takuya Sadahira, Hideo Ueki, Rie Kinoshita, Hitoshi Murata, Ken-Ichi Yamamoto, Junichiro Futami, Yasutomo Nasu, Kazuhiko Ochiai, Hiromi Kumon, Nam-Ho Huh, Masami Watanabe

ONCOLOGY REPORTS 38 ( 2 ) 1108 - 1114 2017年8月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPANDIDOS PUBL LTD

We developed and validated a novel hTERT/CMV promoter element-driven gene expression cassette that can robustly enhance cancer-specific gene expression. The following gene expressional elements were located in tandem within the plasmid construct: [hTERT core promoter, cytomegalovirus (CMV) minimized promoter, RU5' sequence, an inserted gene, BGH polyA, hTERT enhancer]; this is hereafter referred to as the hT/Cm-R-hT construct. Using various human cancer cell lines and normal cells, the cancer-specific transcription of the green fluorescent protein (GFP) gene was examined by western blotting and fluorescence microscopy. Cancer-specific gene expression was robustly achieved in the hT/Cm-R-hT plasmid in comparison to the other control hT/Cm-driven construct. Notably, the expression level of GFP observed in the hT/Cm-RhT-driven construct was superior to that of the control plasmid with the conventional CMV promoter in HEK293 cells, which are known to possess higher hTERT activity than normal cells. We next examined the availability of hT/Cm-R-hT in detecting the target GFP expressing cancer cells from human peripheral blood mononuclear cells (PBMCs). The hT/Cm-R-hT plasmid successfully induced cancer-specific gene expression; the robust expression of GFP was observed in target HeLa cancer cells, whereas GFP was not visibly expressed in normal PBMCs. The plasmid allowed for the selective visualization of viable HeLa cancer cells in mixed cell cultures containing up to 10000-fold more PBMCs. These findings indicate that the hT/Cm-R-hT expressional system is a valuable tool for detecting viable cancer cells mixed with normal cells. The current system can therefore be applied to the in vitro detection of cancer cells that are disseminated in the blood and other types of body fluid in vivo. Since the current system can also be applied to other types of vectors, including virus vectors, this approach using the hTERT promoter-based construct is expected to become a valuable tool for enhancing cancer specific gene expression.

DOI： 10.3892/or.2017.5710

Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3'-side of the transgene 査読

Endy Widya Putranto, Rie Kinoshita, Masami Watanabe, Takuya Sadahira, Hitoshi Murata, Ken-Ichi Yamamoto, Junichiro Futami, Ken Kataoka, Yusuke Inoue, I. Made Winarsa Ruma, I. Wayan Sumardika, Chen Youyi, Miyoko Kubo, Yoshihiko Sakaguchi, Kenji Saito, Yasutomo Nasu, Hiromi Kumon, Nam-Ho Huh, Masakiyo Sakaguchi

ONCOLOGY LETTERS 14 ( 1 ) 1041 - 1048 2017年7月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPANDIDOS PUBL LTD

Reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3) overexpression, induced using an adenovirus (Ad)-REIC, has been revealed to have a dramatic therapeutic effect on multiple types of cancer. To achieve an improved therapeutic effect from Ad-REIC on cancer, our group previously developed an enhanced gene expression system, the C-TSC cassette [cytomegalovirus (CMV)-RU5' located upstream (C); another promoter unit composed of triple tandem promoters, human telomerase reverse transcriptase (hTERT), simian virus 40 and CMV, located downstream of the cDNA (TSC); plus a polyadenylation (polyA) signal]. When applied to the conventional Ad-REIC, this novel system induced the development of an enhanced product, Ad-C-TSC-REIC, which exhibited a noticeable anticancer effect. However, there were difficulties in terms of Ad-C-TSC-REIC productivity in HEK293 cells, which are a widely used donor cell line for viral production. Productivity of Ad-C-TSC-REIC was significantly reduced compared with the conventional Ad-REIC, as the Ad-C-TSC-REIC had a significantly higher ability to induce apoptotic cell death of not only various types of cancer cell, but also HEK293 cells. The present study aimed to overcome this problem by modifying the C-TSC structure, resulting in an improved candidate: A C-T cassette (C: CMV-RU5' located upstream; T: another promoter unit composed of a single hTERT promoter, located downstream of the cDNA plus a polyA signal), which demonstrated gene expression comparable to that of the C-TSC system. The improved adenovirus REIC/Dkk-3 product with the C-T cassette, named Ad-C-T-REIC, exhibited a higher expression level of REIC/Dkk3, similar to that of Ad-C-TSC-REIC. Notably, the vector mitigated the cell death of donor HEK293 cells, resulting in a higher rate of production of its adenovirus. These results indicated that Ad-C-T-REIC has the potential to be a useful tool for application in cancer gene therapy.

DOI： 10.3892/ol.2017.6201

An HNF4α-microRNA-194/192 signaling axis maintains hepatic cell function. 査読

Morimoto A, Kannari M, Tsuchida Y, Sasaki S, Saito C, Matsuta T, Maeda T, Akiyama M, Nakamura T, Sakaguchi M, Nameki N, Gonzalez FJ, Inoue Y

The Journal of biological chemistry 292 ( 25 ) 10574 - 10585 2017年6月

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

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年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：INT INST ANTICANCER RESEARCH

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

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

Masakiyo Sakaguchi

THERANOSTICS 7 ( 10 ) 2649 - 2651 2017年

記述言語：英語出版者・発行元：IVYSPRING INT PUBL

Great progress has been made in in vivo imaging for cancer metastasis. Eisenblaetter et al. developed an innovative S100A8/A9-specific single photon emission computed tomography (SPECT) probe for imaging and succeeded in detecting the metastatic organ favored by the cancer before the cancer arrival. By utilizing this advanced method, researchers have also found that cancer-derived monocytes are the main source of the abundant production of S100A8/A9 in the pre-metastatic area. The CCL2-CCR2 axis is associated with S100A8/A9 production. Clinical establishment of this technique is expected to enable accurate prediction and monitoring of cancer metastasis during therapy.

DOI： 10.7150/thno.19866

平成28年度岡山医学会賞総合研究奨励賞（結城賞）

阪口政清

岡山医学会雑誌 129 ( 2 ) 81 - 83 2017年

Crucial Role of RAGE in Inappropriate Increase of Smooth Muscle Cells From Patients With Pulmonary Arterial Hypertension 査読

Kazufumi Nakamura, Satoshi Akagi, Toshihiro Sarashina, Masakiyo Sakaguchi, Hiromi Matsubara, Hiroshi Ito

CIRCULATION 134 2016年11月

PINK1 Regulation of Mitochondrial Homeostasis and Cell Survival. 査読国際誌

Hitoshi Murata, Ken-ichi Yamamoto, Rie Kinoshita, Ken Kataoka, Nam-ho Huh, Masakiyo Sakaguchi

IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL 52 S8 - S8 2016年6月

Development of a Novel REIC/DKK-3-Encoding Adenoviral Agent: Its Robust and Promising Therapeutic Effects in Pancreatic Cancer 査読

Daisuke Uchida, Hidenori Shiraha, Hironari Kato, Sawahara Hiroaki, Masakiyo Sakaguchi, Masami Watanabe, Yasutomo Nasu, Hiromi Kumon, Hiroyuki Okada

GASTROENTEROLOGY 150 ( 4 ) S294 - S294 2016年4月

Receptor for advanced glycation endproducts modulates leukotriene B-4 receptor 1 signaling 査読

Takako Ichiki, Tomoaki Koga, Toshiaki Okuno, Kazuko Saeki, Yasuhiko Yamamoto, Masakiyo Sakaguchi, Takehiko Yokomizo

FASEB JOURNAL 30 2016年4月

Promising Therapeutic Efficacy of a Novel REIC/Dkk-3 Expressing Adenoviral Vector for Hepatocellular Carcinoma 査読

Sawahara Hiroaki, Hidenori Shiraha, Daisuke Uchida, Masakiyo Sakaguchi, Masami Watanabe, Yasutomo Nasu, Hiromi Kumon, Hiroyuki Okada

GASTROENTEROLOGY 150 ( 4 ) S1153 - S1153 2016年4月

Receptor for advanced glycation endproducts modulates leukotriene B-4 receptor 1 signaling 査読

Takako Ichiki, Tomoaki Koga, Toshiaki Okuno, Kazuko Saeki, Yasuhiko Yamamoto, Masakiyo Sakaguchi, Takehiko Yokomizo

FASEB JOURNAL 30 2016年4月

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

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年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

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

NRF2 Regulates PINK1 Expression under Oxidative Stress Conditions 査読国際誌

Hitoshi Murata, Hitoshi Takamatsu, Sulai Lie, Ken Kataoka, Nam-ho Huh, Masakiyo Sakaguchi

PLoS One 10 ( 11 ) e0142438 2015年11月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PUBLIC LIBRARY SCIENCE

Mutations of the PTEN-induced putative kinase 1 (PINK1) gene are a cause of autosomal recessive forms of Parkinson's disease. Recent studies have revealed that PINK1 is an essential factor for controlling mitochondrial quality, and that it protects cells from oxidative stresses. Although there has been considerable progress in the elucidation of various aspects of PINK1 protein regulation such as activation, stability and degradation, the transcriptional regulation of PINK1 mRNA under stress conditions remains unclear. In this study, we found that nuclear factor (erythroid-derived 2)-like 2 (NRF2), an antioxidant transcription factor, regulates PINK1 expression under oxidative stress conditions. Damaged mitochondria arising from stress conditions induced NRF2-dependent transcription of the PINK1 gene through production of reactive oxygen species (ROS). Either an ROS scavenger or forced expression of KEAP1, a potent inhibitory partner to NRF2, restricted PINK1 expression induced by activated NRF2. Transcriptionally up-regulated PINK1 diminished oxidative stress-associated cell death. The results indicate that PINK1 expression is positively regulated by NRF2 and that the NRF2-PINK1 signaling axis is deeply involved in cell survival.

DOI： 10.1371/journal.pone.0142438

REIC/Dkk-3遺伝子治療の胸部悪性腫瘍に対する免疫刺激を介する抗腫瘍効果

諏澤憲, 枝園和彦, 阪口政清, 渡部昌実, 橋田真輔, 宗淳一, 山本寛斉, 牧祐歩, 佃和憲, 那須保友, 公文裕巳, 三好新一郎, 豊岡伸一

日本癌学会総会記事 74回 J - 1224 2015年10月

T790M変異を含むEGFR遺伝子変異を有する非小細胞肺癌細胞株に対するTAE226の抗腫瘍効果

山本寛斉, 阪口政清, 宗淳一, 佃和憲, 木浦勝行, 猶本良夫, 三好新一郎, 豊岡伸一

日本癌学会総会記事 74回 P - 2193 2015年10月

TAE226, a Bis-Anilino Pyrimidine Compound, Shows Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells including T790M Mutant 査読

Hiromasa Yamamoto, Hiroki Otani, 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

JOURNAL OF THORACIC ONCOLOGY 10 ( 9 ) S311 - S312 2015年9月

Distant Bystander Effect of REIC/Dkk-3 Gene Therapy through Immune System Stimulation in a Murine Model of Thoracic Malignancies 査読

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

JOURNAL OF THORACIC ONCOLOGY 10 ( 9 ) S599 - S599 2015年9月

Anti-tumor effect of afatinib, an irreversible EGFR/HER2 dual inhibitor, in lung cancers harboring HER2 oncogene 査読

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

CANCER RESEARCH 75 2015年8月

Identification of a novel binding protein playing a critical role in HER2 activation in lung cancer cells 査読

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

CANCER RESEARCH 75 2015年8月

Novel germline G660D mutation in HER2 gene detected by whole-exome sequencing can predispose a patient to developing familial lung adenocarcinoma 査読

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

CANCER RESEARCH 74 ( 19 ) 2014年10月

家族性・孤発性肺腺癌におけるHER2膜貫通領域の新規遺伝子変異(Novel functional mutations in the transmembrane domain of HER2 gene in familial and sporadic lung adenocarcinomas)

山本寛斉, 日笠幸一郎, 阪口政清, 枝園和彦, 宗淳一, 市村浩一, 佃和憲, 瀧川奈義夫, 松尾恵太郎, 木浦勝行, 三好新一郎, 松田文彦, 豊岡伸一

日本癌学会総会記事 73回 E - 2012 2014年9月

新規HER2膜貫通部領域遺伝子変異の機能解析(Novel HER2 mutations in transmembrane dmain result in constitutive autophosphorylation of HER2)

諏澤憲, 阪口政清, 山本寛斉, 宗淳一, 牧佑歩, 橋田真輔, 大塚智昭, 渡邉元嗣, 浅野博昭, 佃和憲, 三好新一郎, 豊岡伸一

日本癌学会総会記事 73回 P - 1328 2014年9月

がんマーカー、サイトケラチン19(CK19)機能の新展開 HER2活性化におけるCK19の役割(Critical role of Cytokeratin-19 in an oncogenic activation of HER2)

大塚智昭, 阪口政清, 渡邉元嗣, 諏澤憲, 橋田真輔, 牧佑歩, 山本寛斉, 宗淳一, 浅野博昭, 佃和憲, 三好新一郎, 豊岡伸一

日本癌学会総会記事 73回 E - 2071 2014年9月

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

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年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPANDIDOS PUBL LTD

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

Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene 査読国際誌

Masakiyo Sakaguchi, Masami Watanabe, Rie Kinoshita, Haruki Kaku, Hideo Ueki, Junichiro Futami, Hitoshi Murata, Yusuke Inoue, Shun-Ai Li, Peng Huang, Endy Widya Putranto, I. Made Winarsa Ruma, Yasutomo Nasu, Hiromi Kumon, Nam-ho Huh

MOLECULAR BIOTECHNOLOGY 56 ( 7 ) 621 - 630 2014年7月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：HUMANA PRESS INC

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1 alpha, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5' located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.

DOI： 10.1007/s12033-014-9738-0

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

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

NATURE COMMUNICATIONS 5 3795 2014年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

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

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年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPANDIDOS PUBL LTD

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

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

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 2014年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PUBLIC LIBRARY SCIENCE

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

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

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年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OKAYAMA UNIV MED SCHOOL

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

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

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 2014年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OXFORD UNIV PRESS INC

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

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

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 ) 2014年

掲載種別：研究論文（学術雑誌）出版者・発行元：1

© 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

Histidine-rich glycoprotein prevents septic lethality through neutrophil regulation 査読

Nishibori M, Wake H, Mori S, Liu K, Morioka Y, Teshigawara K, Sakaguchi M, Kuroda K, Takahashi H, Ohtsuka A, Yoshino T, Morimatsu H

Critical Care 1 - 53 2014年

Inhibition of RAGE signaling through the intracellular delivery of inhibitor peptides by PEI cationization 査読

Endy Widya Putranto, Hitoshi Murata, Ken-Ichi Yamamoto, Ken Kataoka, Hidenori Yamada, Jun-Ichiro Futami, Masakiyo Sakaguchi, Nam-Ho Huh

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 32 ( 4 ) 938 - 944 2013年10月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPANDIDOS PUBL LTD

The receptor for advanced glycation end products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobulin superfamily. RAGE is involved in a wide range of inflammatory, degenerative and hyper-proliferative disorders which span over different organs by engaging diverse ligands, including advanced glycation end products, S100 family proteins, high-mobility group protein B1 (HMGB1) and amyloid beta. We previously demonstrated that the cytoplasmic domain of RAGE is phosphorylated upon the binding of ligands, enabling the recruitment of two distinct pairs of adaptor proteins, Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) and myeloid differentiation protein 88 (MyD88). This engagement allows the activation of downstream effector molecules, and thereby mediates a wide variety of cellular processes, such as inflammatory responses, apoptotic cell death, migration and cell growth. Therefore, inhibition of the binding of TIRAP to RAGE may abrogate intracellular signaling from ligand-activated RAGE. In the present study, we developed inhibitor peptides for RAGE signaling (RAGE-I) by mimicking the phosphorylatable cytosolic domain of RAGE. RAGE-I was efficiently delivered into the cells by polyethylenimine (PEI) cationization. We demonstrated that RAGE-I specifically bound to TIRAP and abrogated the activation of Cdc42 induced by ligand-activated RAGE. Furthermore, we were able to reduce neuronal cell death induced by an excess amount of S100B and to inhibit the migration and invasion of glioma cells in vitro. Our results indicate that RAGE-I provides a powerful tool for therapeutics to block RAGE-mediated multiple signaling.

DOI： 10.3892/ijmm.2013.1467

SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria 査読

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

MOLECULAR BIOLOGY OF THE CELL 24 ( 18 ) 2772 - 2784 2013年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC CELL BIOLOGY

Mutations in PTEN-induced putative kinase 1 (PINK1) or parkin cause autosomal recessive forms of Parkinson's disease. Recent work suggests that loss of mitochondrial membrane potential stabilizes PINK1 and that accumulated PINK1 recruits parkin from the cytoplasm to mitochondria for elimination of depolarized mitochondria, which is known as mitophagy. In this study, we find that PINK1 forms a complex with sterile alpha and TIR motif containing 1 (SARM1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), which is important for import of PINK1 in the outer membrane and stabilization of PINK1 on depolarized mitochondria. SARM1, which is known to be an adaptor protein for Toll-like receptor, binds to PINK1 and promotes TRAF6-mediated lysine 63 chain ubiquitination of PINK1 at lysine 433. Down-regulation of SARM1 and TRAF6 abrogates accumulation of PINK1, followed by recruitment of parkin to damaged mitochondria. Some pathogenic mutations of PINK1 reduce the complex formation and ubiquitination. These results indicate that association of PINK1 with SARM1 and TRAF6 is an important step for mitophagy.

DOI： 10.1091/mbc.E13-01-0016

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

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年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPANDIDOS PUBL LTD

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

S100A9 is a novel ligand of EMMPRIN that promotes melanoma metastasis 査読

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年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：1

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

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

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年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPANDIDOS PUBL LTD

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

DIRECT THERAPEUTIC EFFECT OF REIC/DKK-3-ENCODING ADENOVIRAL VECTOR FOR NON-SMALL CELL LUNG CANCER 査読

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

JOURNAL OF THORACIC ONCOLOGY 7 ( 11 ) S463 2012年11月

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

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年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPANDIDOS PUBL LTD

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

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

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

ONCOLOGY LETTERS 3 ( 5 ) 1149 - 1153 2012年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPANDIDOS PUBL LTD

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

非小細胞肺癌に対する新たな個別化治療戦略 REIC/Dkk-3遺伝子治療の可能性

宗淳一, 豊岡伸一, 田中則光, 渡部昌実, 山本寛斉, 阪口政清, 許南浩, 那須保友, 公文裕巳, 三好新一郎

日本呼吸器外科学会雑誌 26 ( 3 ) P17 - 01 2012年4月

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

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

ONCOLOGY REPORTS 27 ( 3 ) 695 - 699 2012年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPANDIDOS PUBL LTD

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

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

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年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

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 査読

Masakiyo Sakaguchi, Nam-ho Huh, Masayoshi Namba

HUMAN CELL TRANSFORMATION: ROLE OF STEM CELLS AND THE MICROENVIRONMENT 720 209 - 215 2012年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER-VERLAG BERLIN

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 overexpressed 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.

DOI： 10.1007/978-1-4614-0254-1_17

S100A11, a dual growth regulator of epidermal keratinocytes 査読

Masakiyo Sakaguchi, Nam-ho Huh

AMINO ACIDS 41 ( 4 ) 797 - 807 2011年10月

記述言語：英語出版者・発行元：SPRINGER

S100A11, a member of the family of S100 proteins, is a dimmer, each monomer of which has two EF-hands. Expression of S100A11 is ubiquitous in various tissues at different levels, with a high expression level in the skin. We have analyzed functions of S100A11 mainly in normal human keratinocytes (NHK) as a model cell system of human epithelial cells. High Ca(2+) and transforming growth factor-beta (TGF-beta), two representative growth suppressors for NHK, need a common S100A11-mediated pathway in addition to unique pathways (NFAT1-mediated pathway for high Ca(2+) and Smad-mediated pathway for TGF-beta) for exhibiting a growth inhibitory effect. S100A11 has another action point for growth suppression in NHK. Annexin A1 (ANXA1) complexed with S100A11 efficiently binds to and inhibits cytosolic phospholipase A2 (cPLA2), the activity of which is needed for the growth of NHK. On exposure of NHK to epidermal growth factor (EGF), ANXA1 is cleaved at 12Trp, and this truncated ANXA1 loses binding capacity to S100A11, resulting in maintenance of an active state of cPLA2. On the other hand, we found that S100A11 is actively secreted by NHK. Extracellular S100A11 acts on NHK to enhance the production of EGF family proteins, resulting in growth stimulation. These findings indicate that S100A11 plays a dual role in growth regulation, being suppressive in cells and being promotive from outside of cells.

DOI： 10.1007/s00726-010-0747-4

悪性胸膜中皮腫に対するマイクロRNA34b/cを用いたアデノウイルス遺伝子治療の可能性(Anti-proliferative effect of microRNA-34b/c adenoviral gene transfer on malignant pleural mesotheliomas)

宗淳一, 豊岡伸一, 久保孝文, 上野剛, 深澤拓也, 阪口政清, 佃和憲, 浅野博昭, 山本寛斉, 許南浩, 三好新一郎

日本癌学会総会記事 70回 267 - 267 2011年9月

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

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年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

The impact of adenoviral gene transfer of microRNA-34b/c on malignant pleural mesotheliomas 査読

Junichi Soh, Shinichi Toyooka, Takafumi Kubo, Takuya Fukazawa, Masakiyo Sakaguchi, Tsuyoshi Ueno, Kazunori Tsukuda, Hiroaki Asano, Hiromasa Yamamoto, Nam-ho Huh, Shinichiro Miyoshi

CANCER RESEARCH 71 2011年4月

マイクロRNA34b/cのメチル化による発現低下は悪性胸膜中皮腫の重要な分子病態である(Epigenetic silencing of microRNA-34b/c plays a pivotal role in pathogenesis of malignant mesothelioma pathogenesis)

久保孝文, 豊岡伸一, 佃和憲, 阪口政清, 深澤拓也, 宗淳一, 浅野博昭, 那須保友, 岸本卓巳, 松井秀樹, 許南浩, 三好新一郎

日本癌学会総会記事 69回 44 - 44 2010年8月

Frequent silencing of tumor suppressive MicroRNA-34b/c by aberrant methylation in malignant mesothelioma 査読

Takafumi Kubo, Shinichi Toyooka, Kazunori Tsukuda, Hiroaki Asano, Masakiyo Sakaguchi, Junichi Soh, Tsuyoshi Ueno, Hiromasa Yamamoto, Masaomi Yamane, Takahiro Oto, Yasutomo Nasu, Hideki Matsui, Nam Ho Huh, Shinichro Miyoshi

CANCER RESEARCH 70 2010年4月

REIC/Dkk-3 stable transfection reduces the malignant phenotype of mouse prostate cancer RM9 cells 査読

Jie Chen, Masami Watanabe, Peng Huang, Masakiyo Sakaguchi, Kazuhiko Ochiai, Yasutomo Nasu, Mamoru Ouchida, Nam-Ho Huh, Kenji Shimizu, Yuji Kashiwakura, Haruki Kaku, Hiromi Kumon

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 24 ( 6 ) 789 - 794 2009年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPANDIDOS PUBL LTD

The reduced expression in immortalized cells (REIC)/Dickkopf (Dkk)-3. a member of the Dkk gene family, is a tumor suppressor in a broad range of cancers. REIC/Dkk-3 transfected stable clones of mouse prostate cancer RM9 cells (RM9-REIC) and the empty vector-transfected control clone cells (RM9-EV) were established. Clones were used to evaluate the anti-cancer effects and a proteomics analysis of REIC/Dkk-3 continuous expression was performed. The RM9-REIC cells show a feeble appearance and the cell membrane shows irregular buds known as blebs. In vitro cell proliferation was significantly suppressed in RM9-REIC clones in comparison to the control. The apoptosis assay was done under standard culture conditions and RM9-REIC showed a higher incidence of apoptosis. The RM9-EV and RM9-REIC cells were orthotopically implanted into a C57BL/6 mouse prostate. After 2 weeks, the tumor growth was significantly inhibited in RM9-REIC cells in comparison to the control. Two-dimensional gel electrophoresis was used to examine the modification of protein expression by the gene transfection. The analysis with mass spectrometry disclosed that expression of peroxiredoxin-l, GST-P1. transgelin-2, MRP-L12, ARD, GRP78 and Sorcin were increased and eEF1A-1 and cyclophilin-40 protein were decreased in RM9-REIC cells. Therefore, REIC/Dkk-3 stable transfectants show a reduction of malignancy in mouse prostate cancer RM9 cells in vitro and in vivo. The result of the proteomics analysis might provide important clues to clarify the anti-cancer molecular mechanism of REIC/Dkk-3 gene transfer.

DOI： 10.3892/ijmm_00000293

S100A11, a dual mediator for growth regulation of normal human keratinocytes 査読

Masakiyo Sakaguchi, Nam-ho Huh

AMINO ACIDS 37 ( 1 ) 77 - 77 2009年7月

Dual tyrosine kinase inhibitor for focal adhesion kinase and insulin-like growth factor-I receptor (TAE226) leads to apoptosis in esophageal cancer by inhibiting AKT-mTOR survival signaling 査読

Huifang Hao, Zhigang Wang, Xiaohong Bao, Nobuyuki Watanabe, Kazufumi Sakurama, Yasuko Tomono, Takuya Fukazawa, Shinji Hatakeyama, Osamu Omori, Seishi Nishitani, Toshiaki Ohara, Munenori Takaoka, Yasuhiro Shirakawa, Tomoki Yamatsuji, Junji Matsuoka, Masakiyo Sakaguchi, Noriaki Tanaka, Yoshio Naomoto

CANCER RESEARCH 69 2009年5月

Adenovirus-mediated REIC/DKK-3 gene transfer prevented mesothelioma tumor progressions in orthotopic mice model 査読

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

JOURNAL OF GENE MEDICINE 10 ( 4 ) 469 - 470 2008年4月

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

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

JOURNAL OF GENE MEDICINE 10 ( 4 ) 454 - 454 2008年4月

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

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年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC CANCER RESEARCH

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

NMR studies of the N-terminal segment of S100C/A11 protein 査読

Takahide Kouno, Mineyuki Mizuguchi, Masakiyo Sakaguchi, Eiichi Makino, Nam-ho Huh, Keiichi Kawano

Peptides 2004, Proceedings 1025 - 1026 2005年

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

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年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PROFESSOR D A SPANDIDOS

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.

Micromechanics of the damage-induced cellular microstructure in single crystal Ni-based superalloys

M. Sakaguchi, M. Okazaki

Acta Metallurgica Sinica (English Letters) 17 ( 4 ) 361 - 368 2004年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

An analytical method to investigate the morphological evolution of the cellular microstructure is explored and proposed. The method is essentially based on the Eshelby's micromechanics theory, and it is extended so as to be applied for a material system containing inclusions with high volume fraction, by employing the average stress field approximation by Mori and Tanaka. The proposed method enables us to discuss a stable shape of precipitate in the material system, which must be influenced by many factors: e.g., volume fraction of precipitate
Young's modulus ratio and lattice misfit between matrix and precipitate
external stress field in multiaxial state
and heterogeneity of plastic strain between matrix and precipitate. A series of numerical calculations were summarized on stable shape maps. The application of the method to predict the γ′ rafting in superalloys during creep showed that the heterogeneity of plastic strain between matrix and precipitates may play a significant role in the shape stability of the precipitate. Furthermore, it was shown that the method was successfully applied to estimate the morphology of the cellular microstructure formed in CMSX-4 single crystal Ni-based superalloy.

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

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

CANCER RESEARCH 64 ( 12 ) 4155 - 4161 2004年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC CANCER RESEARCH

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

Differential expression of S100C in thyroid lesions 査読

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年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

PKC alpha mediates TGF beta-induced growth inhibition of human keratinocytes via phosphorylation of S100C/A11 査読

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

JOURNAL OF CELL BIOLOGY 164 ( 7 ) 979 - 984 2004年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ROCKEFELLER UNIV PRESS

Growth regulation of epithelial cells is of major concern because most human cancers arise from them. We demonstrated previously a novel signal pathway involving S100C/A11 for high Ca2+-induced growth inhibition of normal human keratinocytes (Sakaguchi, M., M. Miyazaki, M. Takaishi, Y. Sakaguchi, E. Makino, N. Kataoka, H. Yamada, M. Namba, and N.H. Huh. 2003.J. Cell Biol. 163:825-835). This paper addresses a question whether transforming growth factor beta (TGFbeta) shares the pathway with high Ca2+. On exposure of the cells to TGFbeta1, S100C/A11 was phosphorylated, bound to nucleolin, and transferred to the nucleus, resulting in induction of p21(WAF1/CIP1) and p15(INK4B) through activation of Sp1. Protein kinase C alpha (PKCalpha) was shown to phosphorylate. (10)Thr of S100C/A11, which is a critical event for the signal transduction. The TGFbeta1-induced growth inhibition was almost completely mitigated when PKCalpha activity was blocked or when S100C/A11 was functionally sequestered. These results indicate that, in addition to the well-characterized Smad-mediated pathway, the PKCalpha-S100C/A11-mediated pathway is involved in and essential for the growth inhibition of normal human keratinocytes cells by TGFbeta1.

DOI： 10.1083/jcb.200312041

Lentiviral vector: A useful tool for transduction of human liver endothelial cells 査読

T Totsugawa, N Kobayashi, M Maruyama, Y Kosaka, T Okitsu, T Arata, M Sakaguchi, T Ueda, Y Kurabayashi, N Tanaka

ASAIO JOURNAL 49 ( 6 ) 635 - 640 2003年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：LIPPINCOTT WILLIAMS & WILKINS

Endothelial cells play multiple roles in pathophysiologic processes and are increasingly being recognized as target cells of gene therapy. Lentiviral vectors derived from human immunodeficiency virus type 1 have an ability to infect both dividing and nondividing cells and currently receive a great deal of attention as an innovative tool for transduction of target cells. The purpose of the present work was to evaluate the efficacy of a lentiviral vector for transducing human liver endothelial cells (HLECs) in vitro. For the present study, a pseudotyped lentiviral vector encoding a green fluorescent protein (GFP) gene, LtV-GFP, was generated by means of FuGENE 6 method and allowed to infect HLECs. Approximately 95% of HLECs were positive for GFP expression after LtV-GFP infection at a multiplicity of infection of 10. Notably, LtV-GFP transduced HLECs had stable and long term GFP expression, showed gene expression of endothelial markers including CD 34, factor VIII, flt-1, KDR/flk-1 and HGF, and maintained in vitro angiogenic potential in a Matrigel assay to the same extent as primarily cultured HLECs. These findings provide evidence that lentivirus based gene delivery is an efficient tool for transduction of endothelial cells that could be considered for cell and gene therapies and hybrid artificial organs.

DOI： 10.1097/01.MAT.0000093747.89681.4C

BRPK, a novel protein kinase showing increased expression in mouse cancer cell lines with higher metastatic potential 査読

A Nakajima, K Kataoka, M Hong, M Sakaguchi, N Huh

CANCER LETTERS 201 ( 2 ) 195 - 201 2003年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCI IRELAND LTD

A novel protein kinase named BRPK was isolated and partially characterized. BRPK was expressed at a higher level in three carcinoma cell lines with higher metastatic potential. Mouse and human BRPK cDNAs are well conserved and encode 580 and 581 amino acids, respectively. BRPK has a serine/threonine-type protein kinase domain, and the recombinant proteins of BRPK were capable of autophosphorylation. The results of a comparative sequence analysis indicated a possible link of BRPK to BRAP2. BRAP2 is known to bind the nuclear localization signal of BRCA1. We cloned mouse BRAP2 cDNA and showed the presence of isoforms. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/S0304-3835(03)00443-9

Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers 査読

T Watanabe, N Shibata, KA Westerman, T Okitsu, JE Allain, M Sakaguchi, T Totsugawa, M Maruyama, T Matsumura, H Noguchi, S Yamamoto, M Hikida, A Ohmori, M Reth, A Weber, N Tanaka, P Leboulch, N Kobayashi

TRANSPLANTATION 75 ( 11 ) 1873 - 1880 2003年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：LIPPINCOTT WILLIAMS & WILKINS

Background. Maintenance of liver-specific functions has been shown to be stabilized by co-cultivation of hepatocytes with hepatic stellate cells (HSC). Because the limited lifespan of human HSC is a major hurdle to their use, the authors report here the amplification of human HSC populations in vitro by retroviral transfer of human telomerase reverse transcriptase (hTERT).
Methods. Human HSC strain LI 90 cells were transduced with a retroviral vector SSR#197 expressing hTERT and green fluorescent protein (GFP) cDNA flanked by a pair of loxP. TWNT-1, one of SSR#197-immortalized HSC, was characterized. Differentiated liver functions were evaluated in an immortalized human hepatocyte NKNT-3-TWNT-1 co-culture system.
Results. TWNT-1 cells showed differential functions of HSC, including uptake of acetylated low-density lipoproteins and synthesis of collagen type I and hepatocyte growth factor. Efficient excision of the retro-virally transferred hTERT and GFP cDNAs was achieved by TAT-mediated expression of the Cre recombinase and subsequent GFP-negative cell sorting. When co-cultured with TWNT-1 cells, NKNT-3 increased protein expression of the detoxifying cytochrome P450-associated protein isoenzymes 3A4 and 2C9 and urea synthesis.
Conclusions. TWNT-1 cells could be valuable in the study of integrated liver functions and contribute to the optimization of liver cell therapies and bioartificial livers.

DOI： 10.1097/01.TP.0000064621.50907.A6

Involvement of arachidonic acid in nonimmunologic production of superoxide in mast cells 査読

M Sakaguchi, N Fukuishi, K Teramoto, M Miyazaki, NH Huh, M Namba, M Akagi

INTERNATIONAL ARCHIVES OF ALLERGY AND IMMUNOLOGY 130 ( 4 ) 288 - 299 2003年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：KARGER

Background: A number of different molecules are known to be involved in the signal pathway to release histamine from mast cells, among which arachidonic acid (AA) is one of the key mediators. On the other hand, we found that the application of compound 48/80, a typical histamine liberator, generated superoxide in mast cells. In the present study, we investigated the mechanism of superoxide production in mast cells with respect to AA signaling in conjunction with a fine structural analysis. Methods: Superoxide production was monitored by chemiluminescence in rat peritoneal mast cells and their subfractions after various treatments. For scanning electron micrography, the conditions for fixation and freeze-fracture were optimized to get natural fine images. Results: Compound 48/80 induced superoxide production in the isolated mast cells and some of their subfractions possibly through intracellular increase in Ca2+ concentration, activation of cytosolic phospholipase A(2), and release of AA. Discussion: The present results indicate the critical role of AA in the signal pathway to generate superoxide from mast cells in response to compound 48/80. Copyright (C) 2003 S. Karger AG, Basel.

DOI： 10.1159/000070216

Establishment of an immortalized human hepatic stellate cell line to develop antifibrotic therapies 査読

Norikuni Shibata, Takamasa Watanabe, Teru Okitsu, Masakiyo Sakaguchi, Michihiko Takesue, Takemi Kunieda, Kenji Omoto, Shinichiro Yamamoto, Noriaki Tanaka, Naoya Kobayashi

Cell Transplantation 12 ( 5 ) 499 - 507 2003年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cognizant Communication Corporation

Because human hepatic stellate cells (HSCs) perform a crucial role in the progress of hepatic fibrosis, it is of great value to establish an immortalized human cell line that exhibits HSC characteristics and grows well in tissue cultures for the development of antifibrotic therapies. Thus, we engineered an immortalized human hepatic stellate cell (HSC) line TWNT-4 by retrovirally inducing human telomerase reverse transcriptase (hTERT) into LI 90 cells established from a human liver mesenchymal tumor. Parental LI 90 entered replicative senescence, whereas TWNT-4 showed telomerase activity and proliferated for more than population doubling level (PDL) 200 without any crisis. TWNT-4 expressed platelet-derived growth factor-β receptor (PDGF-βR), α-smooth muscle actin (α-SMA), and type I collagen (α1) and was considered to be an activated form of HSCs. Treatment of TWNT-4 cells with either 100 U/ml of IFN-γ or 1 ng/ml of rapamycin (Rapa) for 14 days led to lower expression of type I collagen (α1) at RNA and protein levels. Exposure of TWNT-4 cells to both of IFN-γ (10 U/ml) and Rapa (0.1 ng/ml) for 14 days effectively decreased the expression of type I collagen (α1), PDGF-βR, and α-SMA expression and suppressed TGF-β1 secretion of TWNT-4 cells. We successfully induced apoptosis by transducing TNF-related apoptosis-inducing ligand (TRAIL) into TWNT-4 cells using adenovirus vectors Ad/GT-TRAIL and Ad/PGK-GV-17. These findings suggested that immortalized activated HSC line TWNT-4 would be a useful means to develop antifibrotic therapies.

DOI： 10.3727/000000003108747064

Active expression of p21 facilitates differentiation of immortalized human hepatocytes 査読

N. Kobayashi, T. Kunieda, M. Sakaguchi, T. Okitsu, T. Totsugawa, M. Maruyama, Y. Kosaka, M. Takesue, N. Shibata, N. Tanaka

Transplantation Proceedings 35 ( 1 ) 433 - 434 2003年

Maintenance of cold-preserved porcine hepatocyte function with UW solution and ascorbic acid-2 glucoside

M Takesue, M Maruyama, N Shibata, T Kunieda, T Okitsu, M Sakaguchi, T Totsugawa, Y Kosaka, A Arata, H Ikeda, J Matsuoka, T Oyama, M Kodama, K Ohmoto, S Yamamoto, Y Kurabayashi, Yamamoto, I, N Tanaka, N Kobayashi

CELL TRANSPLANTATION 12 ( 6 ) 599 - 606 2003年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SAGE PUBLICATIONS INC

Normal human hepatocytes are an ideal source of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, but availability of human donor livers for liver cell isolation is severely limited. To effectively utilize scarce donor organs for cell therapies, it is of extreme importance to establish an efficient isolation technique and an effective cold preservation solution for transportation of isolated cells. A lateral segment of the liver was surgically resected from pigs weighing 10 kg and a four-step collagenase and dispase digestion was conducted. Isolated hepatocytes were subjected to 8-h cold storage on ice. The following preservation solutions were tested: 1) University of Wisconsin (UW) solution, 2) UW with 100 mug/ml of ascorbic acid-2 glucoside (AA2G), 3) 100% fetal bovine serum (FBS), and 4) Dulbecco's modified Eagle's medium (DMEM) supplemented with 100% FBS. The mean viability of porcine hepatocytes was 95.5 +/- 2.5% when isolated in three independent experiments. Viability, plating efficiency, membrane stability, and ammonia metabolic capacity of cold-preserved hepatocytes were significantly better maintained by the use of UW solution. When AA2G (100 mug/ml) was combined with UW solution, such parameters were further improved. It was explained by inhibition of caspase-3 activation and retention of ATP at high levels of hepatocytes preserved with UW solution containing AA2G. The present work demonstrates that a combination of UW solution with AA2G (100 mug/ml) would be a useful cold preservation means for the development of cell therapies.

DOI： 10.3727/000000003108747208

Improved conditions to induce hepatocytes from rat bone marrow cells in culture 査読

M Miyazaki, Akiyama, I, M Sakaguchi, E Nakashima, M Okada, K Kataoka, NH Huh

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 298 ( 1 ) 24 - 30 2002年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

Recent studies have revealed that bone marrow cells can develop into hepatocytes by in vivo transplantation under certain circumstances. However, little is known about the mechanism of bone marrow cell differentiation into hepatocytes. It is important to determine suitable culture conditions in which bone marrow cells will be differentiated into hepatocytes not only for understanding differentiation mechanisms but also for efficient amplification of hepatocyte-progenitor cells of bone marrow origin, this being a prerequisite for potential therapeutic use. In the present study, we found that hepatocyte growth factor (HGF) receptor (c-Met)- and a-fetoprotein-expressing cells were present in adult rat bone marrow. We also found that these cells also express hematopoietic stem cell markers, such as CD34, Thy-1, and c-Kit. Using an HGM medium with HGF and EGF, we succeeded in propagating hepatocyte-like cells induced from adult rat bone marrow in culture. These cells were immunocytochemically stained for albumin. By RT-PCR analysis of cultures containing the hepatocyte-like cells, we detected mRNAs of tryptophan-2,3-dioxygenase and tyrosine aminotransferase, markers of hepatocytes at a terminal differentiation stage. The present culture therefore can be a useful resource for cell transplantation therapy for liver diseases. (C) 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(02)02340-9

Localization of S100C immunoreactivity in various human tissues 査読

A Kondo, M Sakaguchi, E Makino, M Namba, S Okada, NH Huh

ACTA MEDICA OKAYAMA 56 ( 1 ) 31 - 34 2002年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OKAYAMA UNIV MED SCHOOL

Using 2-dimensional gel electrophoresis, we previously demonstrated that the S100C protein remarkably decreased after immortalization of normal human fibroblasts, and that this protein caused growth inhibition of human tumor cells when forcibly expressed in these cells, suggesting that S100C plays a significant role in tumor suppression. The present study was carried out to determine what type of human tissues express S100C protein,, and, subsequently, whether the S100C content in these tissues changes after normal cells have been transformed into cancer cells. We found that ductal cells in various tissues were positively stained with the S100C protein. In comparison, epithelial cells in digestive organs such as, the stomach, small intestine, and colon were not stained as strongly. When 14 pairs of human normal and cancerous tissues were stained with the antibody, decreases in the staining levels of S100C were observed in 6 kinds of:cancerous tissues-from the bronchus, mammary duct, renal tubule, prostate, uterus, and testis-in comparison with staining in their normal counterparts. These results suggest that S100C is a new tumor marker protein, the expression of which significantly decreases after malignant transformation of human tissues.

Antiproliferative activity of REIC/Dkk-3 and its significant down-regulation in non-small-cell lung carcinomas 査読

T Tsuji, Nozaki, I, M Miyazaki, M Sakaguchi, H Pu, Y Hamazaki, O Iijima, M Namba

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 289 ( 1 ) 257 - 263 2001年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC

We recently reported the cloning of the REIC/Dkk-3 gene, whose expression was shown to be downregulated in many human immortalized and tumor-derived cell lines [T. Tsuji et al. (2000) Biochem. Biophys. Res. Commun. 268, 20-24]. In the present study, we demonstrated that expression of the exogenous REIC/Dkk-3 gene in tumor cells inhibited cell growth. Furthermore, the level of REIC/Dkk-3 mRNA in normal human cells was lowest in the late G, phase during the cell cycle. Then we found that the expression of REIC/Dkk-3 was significantly down-regulated in surgically resected non-small-cell lung carcinomas. We determined the REIC/Dkk-3 locus on chromosome 11p15, where loss of heterozygosity has frequently been observed in human tumors. These findings indicate that REIC/Dkk-3 may function as a tumor suppressor. (C) 2001 Academic Press.

Up-regulation of S100C in normal human fibroblasts in the process of aging in vitro 査読

M Sakaguchi, M Miyazaki, T Kondo, M Namba

EXPERIMENTAL GERONTOLOGY 36 ( 8 ) 1317 - 1325 2001年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD

S100 proteins belonging to the EF-hand Ca2+ -binding protein family regulate a variety of cellular processes via interaction with different target proteins. Several diseases, including cancer and Alzheimer's disease, are related to a disorder of multifunctional S100 proteins, which are expressed in cell- and tissue-specific manners. We previously demonstrated that S100C could move to and accumulate in the nuclei of normal human fibroblasts but not in the nuclei of immortalized and neoplastic cells. In addition, we found that its nuclear accumulation resulted in suppression of DNA synthesis in normal cells at a confluent stage. In the present study, we investigated whether S100C was associated with cellular senescence in vitro. We found that S100C expression increased in normal human fibroblasts in the process of aging in culture and was accompanied by accumulation of its protein in the nuclei of senescent fibroblasts. In addition, the nuclear accumulation of S100C increased expression of a cyclin-dependent kinase inhibitor p21 (Sdil), a strong inhibitor of cell growth. These findings suggest that an increase in the cells having nuclear accumulation of S100C is closely related to the process of cellular senescence of normal human fibroblasts. (C) 2001 Elsevier Science Inc. All rights reserved.

Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV-10 LT 査読

KI Fukaya, S Asahi, S Nagamori, M Sakaguchi, C Gao, M Miyazaki, M Namba

IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL 37 ( 5 ) 266 - 269 2001年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC IN VITRO BIOLOGY

Immortalized human hepatocytes that can retain functions of drug-metabolizing enzymes would be useful for medical and pharmacological studies and for constructing an artificial liver. The aim of this study wa. to establish immortalized human hepatocyte lines having differentiated liver-specific functions. pSVneo deoxyribonucleic acid, which contains large and small T genes in the early region of simian virus 40, was introduced into hepatocytes that had been obtained from the liver of a 21-wk-old fetus. Neomycin-resistant immortalized colonies were cloned and expanded to mass cultures to examine hepatic functions. Cells were cultured in a chemically defined serum-free medium, ASF101, which contains no peptides other than recombinant human transferrin and insulin. As a result, air immortal human hepatocyte cell line (OUMS-29) having li er-specific functions was established from one of the 13 clones. Expression of CYP 1A1 arid 1A2 messenger ribonucleic acid by the cells was induced by treatment with benz[a]pyrene, 3-methylcholanthrene, and benz[a]anthracene. OUMS-29 cells had both the polycyclic aromatic hydrocarbon receptor (AhR) arid AhR nuclear translocator. Consequently, 7-ethoxyresorufin deethylase activity of the cells was induced time- and dose-dependent by these polycyclic aromatic hydrocarbons. This cell line is expected to he instrumental as an alternative method in animal experiments for studying hepatocarcinogenesis. drug metabolisms of liver cells, and hepatic toxicology,.

Overexpression of platelet-derived growth factor B and downregulation of PDGF-receptor alpha in human immortalized fibroblasts 査読

C Gao, M Miyazaki, T Kondo, T Tsuji, M Sakaguchi, M Namba

INTERNATIONAL JOURNAL OF ONCOLOGY 18 ( 4 ) 871 - 875 2001年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PROFESSOR D A SPANDIDOS

Since immortalization of cells is critical in multistep carcinogenesis, efforts should be made to elucidate the mechanisms of the immortalization. To determine whether platelet-derived growth factor (PDGF) signal pathways play a role in immortalization of cells, we compared mRNA expressions of PDGFs and their receptors in three immortalized human fibroblast cell lines (SUSM-1, OUMS-24F, and KMST-6) with their normal parent cells. As a result, mRNA expression of PDGF-B (oncogene: c-sis) was upregulated in these immortalized cells. Unexpectedly, the expression of alpha- and beta -PDGF receptor genes was downregulated. PDGFR-alpha mRNA was remarkably decreased. When exogenous PDGFR-a was expressed transiently in the KMST-6 cells, the morphology of the cells resembled that of normal cells. These results suggest that the overexpression of PDGF-B (c-sis) and downregulation of PDGFR-alpha are related to the phenotypic characteristics of immortalized human cells.

Two-dimensional gel electrophoretic studies on the cellular aging: accumulation of alpha-2-macroglobulin in human fibroblasts with aging

T. Kondo, M. Sakaguchi, M. Namba

Experimental Gerontology 36 ( 3 ) 487 - 495 2001年3月

Identification of a phosphoprotein that is downregulated in immortalized human fibroblasts 査読

M Sakaguchi, M Miyazaki, T Kondo, T Tsuji, H Kouchi, M Namba

ELECTROPHORESIS 22 ( 1 ) 155 - 160 2001年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-V C H VERLAG GMBH

Many lines of evidence indicate that the immortalization step is critical for the neoplastic transformation of normal human cells. Once normal human cells have been immortalized, they are relatively easily transformed into neoplastic cells. In order to understand these phenomena, patterns of protein phosphorylation in proliferating normal human fibroblast cell strains and their immortalized cell lines were compared by using two-dimensional polyacrylamide gel electrophoresis. It was found that the expression and phosphorylation levels of the human heat shock protein 27 (HSP27) were predominantly downregulated in the immortalized cells compared with those in their normal counterparts. In the normal cells, HSP27 expression and phosphorylation were markedly increased by physiological and nonphysiological stresses, such as serum addition, treatment with a carcinogenic agent like 4-nitroquinoline-1-oxide, and a high osmotic pressure. This may be a normal defense against acute changes of cellular environment and cytotoxic effects. However, these stresses had no effects on the expression and phosphorylation of HSP27 in the immortalized cells. These results suggest that an abnormal regulation of HSP27 expression and phosphorylation may be one of the reasons for easy neoplastic transformation of the immortalized cells by the treatment with carcinogenic agents.

Loss of nuclear localization of the S100C protein in immorialized human fibroblasts 査読

M Sakaguchi, H Yamada, T Tsuji, Y Inoue, M Miyazaki, T Tanaka, M Namba

RADIATION RESEARCH 155 ( 1 ) 208 - 214 2001年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：RADIATION RESEARCH SOC

It is well known that cancer develops through a multistep process, lit vitro transformation studies of normal human cells have shown that the immortalization step is critical for neoplastic transformation of cells. Furthermore, studies of cell fusion between normal and immortalized cells have indicated that the normal phenotype is dominant and the immortal phenotype is recessive. Thus we looked for cellular proteins that were down-regulated in immortalized human cells by two-dimensional gel electrophoresis to elucidate the mechanisms of immortalization of human cells. We found that the S100C protein was down-regulated in immortalized cells. This protein was localized in the cytoplasm of cells at the semiconfluent stage, while at the confluent stage it moved into the nuclei of normal cells but not into those of immortalized cells. Microinjection of an S100C antibody into normal confluent cells diminished the level of nuclear S100C protein, resulting in DNA synthesis. Taken together, loss of nuclear localization of the S100C protein, which may be related to DNA synthesis, is thought to be one of the mechanisms of immortalization, (C) 2001 by Radiation Research Society.

Hepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro 査読

SH Oh, M Miyazaki, H Kouchi, Y Inoue, M Sakaguchi, T Tsuji, N Shima, K Higashio, M Namba

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 279 ( 2 ) 500 - 504 2000年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC

Bone marrow (BM) cells originally include alpha-fetoprotein (AFP)- and c-Met [a receptor for hepatocyte growth factor (HGF)]-expressing cells. In vitro treatment of BM cells with HGF induced albumin-expressing hepatocyte-like cells. Furthermore, those hepatocyte-like cells expressed cytokeratins 8 and 18, which are typically expressed in normal adult hepatocytes. These findings demonstrate that BM cells include AFP-expressing hepatic progenitor cells that can be differentiated into hepatocytes by HGF in culture, indicating that such cultures are useful resources for cell transplantation therapy for liver diseases. (C) 2000 Academic Press.

DOI： 10.1006/bbrc.2000.3985

Treatment of surgically induced acute liver failure with transplantation of highly differentiated immortalized human hepatocytes 査読

N Kobayashi, M Miyazaki, K Fukaya, Y Inoue, M Sakaguchi, H Noguchi, T Matsumura, T Watanabe, T Totsugawa, N Tanaka, M Namba

CELL TRANSPLANTATION 9 ( 5 ) 733 - 735 2000年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：COGNIZANT COMMUNICATION CORP

Primary human hepatocytes are an ideal source of hepatic function in bioartficial liver (BAL), but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, primary human fetal hepatocytes were immortalized using simian virus 40 large T antigen. One of the immortal cell lines, OUMS-29, showed highly differentiated liver functions. Intrasplenic transplantation of OUMS-29 cells protected 90% hepatectomized rats from hyperammonemia and significantly prolonged their survival. Essentially unlimited availability of OUMS-29 cells supports their clinical use for BAL treatment.

Relationship between contact inhibition and intranuclear S100C of normal human fibroblasts 査読

M Sakaguchi, M Miyazaki, Y Inoue, T Tsuji, H Kouchi, T Tanaka, H Yamada, M Namba

JOURNAL OF CELL BIOLOGY 149 ( 6 ) 1193 - 1206 2000年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ROCKEFELLER UNIV PRESS

Many lines of evidence indicate that neoplastic transformation of cells occurs by a multistep process. For neoplastic transformation of normal human cells, they must be first immortalized and then be converted into neoplastic cells. It is well known that the immortalization is a critical step for the neoplastic transformation of cells and that the immortal phenotype is recessive. Thus, we investigated proteins downregulated in immortalized cells by two-dimensional gel electrophoresis. As a result, S100C, a Ca2+-binding protein, was dramatically downregulated in immortalized human fibroblasts compared with their normal counterparts. When the cells reached confluence, S100C was phosphorylated on threonine 10. Then the phosphorylated S100C moved to and accumulated in the nuclei of normal cells, whereas in immortalized cells it was not phosphorylated and remained in the cytoplasm. Microinjection of the anti-S100C antibody into normal confluent quiescent cells induced DNA synthesis. Furthermore, when exogenous S100C was compelled to localize in the nuclei of HeLa cells, their DNA synthesis was remarkably inhibited with increase in cyclin-dependent kinase inhibitors such as p16(Ink4a) and p21(Waf1) These data indicate the possible involvement of nuclear S100C in the contact inhibition of cell growth.

In vitro aging research in Japan 査読

T Tsuji, M Miyazaki, M Sakaguchi, M Namba

EXPERIMENTAL GERONTOLOGY 35 ( 3 ) 291 - 298 2000年5月

Establishment of a highly differentiated immortalized human hepatocyte cell line as a source of hepatic function in the bioartificial liver 査読

N Kobayashi, M Miyazaki, K Fukaya, Y Inoue, M Sakaguchi, H Noguchi, N Tanaka, M Namba

TRANSPLANTATION PROCEEDINGS 32 ( 2 ) 237 - 241 2000年3月

Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes 査読

N Kobayashi, T Fujiwara, KA Westerman, Y Inoue, M Sakaguchi, H Noguchi, M Miyazaki, J Cai, N Tanaka, IJ Fox, P Leboulch

SCIENCE 287 ( 5456 ) 1258 - 1262 2000年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC ADVANCEMENT SCIENCE

Because of a critical shortage in suitable organs, many patients with terminal liver disease die each year before liver transplantation can be performed. Transplantation of isolated hepatocytes has been proposed for the temporary metabolic support of patients awaiting liver transplantation or spontaneous reversion of their liver disease. A major limitation of this form of therapy is the present inability to isolate an adequate number of transplantable hepatocytes. A highly differentiated cell line, NKNT-3, was generated by retroviral transfer in normal primary adult human hepatocytes of an immortalizing gene that can be subsequently and completely excised by Cre/Lox site-specific recombination. When transplanted into the spleen of rats under transient immunosuppression, reversibly immortalized NKNT-3 cells provided life-saving metabolic support during acute liver failure induced by 90% hepatectomy.

A REIC gene shows down-regulation in human immortalized cells and human tumor-derived cell lines 査読

T Tsuji, M Miyazaki, M Sakaguchi, Y Inoue, M Namba

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 268 ( 1 ) 20 - 24 2000年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC

Normal human cells stop proliferation after a certain number of cell divisions. This phenomenon is called cellular aging. The fact that the senescence phenotype is dominant and the immortal one is recessive indicates that immortalization of human cells may be caused by loss of functions of certain genes in normal cells. Based on this evidence, several cDNA clones whose expression was down-regulated during the immortalization process of human cells were isolated by the representative difference analysis (RDA) system in our laboratory. One of them, which was named REIC, was expressed to a lower degree in three human immortalized cell lines as compared with their parental normal counterparts, In addition, the expression of REIC was markedly lower in eight human tumor-derived cell lines (Hep3B and HuH-7 hepatocellular carcinomas, HuH-6 Clone 5 hepatoblastoma, HuCCT-1 cholangiocarcinoma, A549 lung cancer, HaCaT immortalized keratinocyte, HeLa cervical carcinoma, and Saos-2 osteosarcoma). In contrast, among the human tissues examined, the heart and brain, which contain a large number of post-mitotic cells, showed the highest expression of REIC. The full-length REIC cDNA revealed that the predicted protein is 350 amino acids in length and possesses coiled-coil tertiary structures in each of the amino- and carboxyl-termini. Furthermore, a search of the protein database revealed a match of this gene product with Dkk-3, which is a novel inhibitor of Wnt oncogene. These results indicate that the REIC cloned by us may function as a tumor suppressor. (C) 2000 Academic Press.

Transplantation of highly differentiated immortalized human hepatocytes to treat acute liver failure 査読

N Kobayashi, M Miyazaki, K Fukaya, Y Inoue, M Sakaguchi, T Uemura, H Noguchi, A Kondo, N Tanaka, M Namba

TRANSPLANTATION 69 ( 2 ) 202 - 207 2000年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：LIPPINCOTT WILLIAMS & WILKINS

Background. Temporary support of a damaged liver by a bioartificial liver (BAL) devise is a promising approach for the treatment of acute liver failure. Although human primary hepatocytes are an ideal source of hepatic function in BAL, shortage of human livers available for hepatocyte isolation is the limiting factor for the use of this modality. A clonal human hepatocyte cell line that can grow economically in culture and exhibit liver-specific functions should be an attractive solution to this problem.
Methods. To test this alternative, primary human fetal hepatocytes were immortalized using Simian virus 40 large T antigen. To investigate the potential of the immortalized cells for BAL, we transplanted the cells into the spleen of adult rats and performed a 90% hepatectomy 12 hr later.
Results, One of the cloned human liver cell lines, OUMS-29, showed highly differentiated liver functions. Intrasplenic transplanting of 20x10(6) OUMS-29 cells protected the animals from hyperammonemia and the associated hepatic encephalopathy, Survival was significantly prolonged in 90% of hepatectomized rats receiving GUMS-29 cells.
Conclusions. A highly differentiated immortalized human hepatocyte cell line, OUMS-29, was able to provide metabolic support during acute liver failure induced by 90% hepatectomy in rats. Essentially unlimited availability of OUMS-29 cells may be clinically useful for BAL treatment.

Establishment of a human hepatoma cell line, HLE/2E1, suitable for detection of P450 2E1-related cytotoxicity 査読

Isao Nozaki, Toshiya Tsuji, Masakiyo Sakaguchi, Yusuke Inoue, Ryuji Hirai, Akio Andou, Masahiro Miyazaki, Nobuyoshi Shimizu, Masayoshi Namba

In Vitro Cellular and Developmental Biology - Animal 36 ( 9 ) 566 - 570 2000年

記述言語：英語掲載種別：研究論文（学術雑誌）

By transfection of an expression vector of human cytochrome P450 2E1 (CYP2E1) into a human hepatoma cell line (HLE), a new cell line (HLE/2E1) that stably expresses activity of CYP2E1 has been established. The HLE/2E1 cell line expressed a higher level of CYP2E1 messenger ribonucleic acid than did the mother HLE cell line. CYP2E1 enzyme activity determined by a p-nitrophenol oxidation assay was also higher in HLE/2E1 cells than in HLE cells. In addition, the enzyme activity of the HLE/2E1 cells was increased by ethanol treatment. Exposure to acetaminophen (APAP) or buthionine sulfoximine (BSO) caused a greater decrease in viability of the HLE/2E1 cells than that of the HLE cells, as determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The cytotoxicity of APAP or BSO to HLE/2E1 cells was inhibited by the addition of ethanol or vitamin E. However, the cytotoxicity of both APAP and BSO was enhanced by 24-h preincubation of HLE/2E1 cells with ethanol. These results show that this cell line provides a useful model for studying catalytic properties of CYP2E1 and cytotoxic mechanisms of chemicals metabolized by CYP2E1.

DOI： 10.1007/BF02577524

Manumycin A, inhibitor of ras farnesyltransferase, inhibits proliferation and migration of rat vascular smooth muscle cells 査読

H Kouchi, K Nakamura, K Fushimi, M Sakaguchi, M Miyazaki, T Ohe, M Namba

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 264 ( 3 ) 915 - 920 1999年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC

Restenosis after angioplasty is thought to be caused by proliferation and migration of vascular smooth muscle cells (VSMCs), and it is a most serious problem in medical treatment. A low dose (50 ng/ml) of manumycin A, an inhibitor of p21(ras) (ras) farnesylation, significantly inhibited proliferation of rat VSMCs stimulated by the platelet-derived growth factor (PDGF). The mitoinhibitory effect of manumycin A was dose- and time-dependent but was independent of cell density. Western blot analysis showed that manumycin A reduced the amount of functional ras localized at the cytoplasmic membrane and inhibited the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK). Manumycin A also inhibited VSMC migration and disorganized alpha actin fibers, as shown by immnofluorecence staining. These results indicate that the interruption of the ras/MAPK signal transduction pathway and the disorganization of alpha actin fibers are the main cause of manumycin A inhibition of VSMC proliferation and migration induced by PDGF. (C) 1999 Academic Press.

Transferrin synthesized in cultured human fibroblasts is associated with tubulins and has iron binding capacity 査読

Masakiyo Sakaguchi, Tadashi Kondo, Hong Pu, Masayoshi Namba

Cell Structure and Function 24 ( 1 ) 5 - 9 1999年

記述言語：英語掲載種別：研究論文（学術雑誌）

In a previous report, using immunocytochemical and fluorescence-labeling techniques, we demonstrated that transferrin is synthesized in cultured human fibroblasts and that it is associated with tubulins in the cells. These morphological findings led us to attempt to elaborate those issues in more detail by biochemical methods. In this report, we were able to prove the association of transferrin produced in cells with tubulins. In addition, the transferrin associated with tubulins was found to bind to iron. These results suggest that endogenous transferrin plays a role in preventing damage caused by free radicals which can be induced by the interaction of iron with the hydrogen peroxide produced in cells.

DOI： 10.1247/csf.24.5

Characteristics of intracellular transferrin produced by human fibroblasts: Its posttranscriptional regulation and association with tubulin 査読

Tadashi Kondo, Masakiyo Sakaguchi, Masayoshi Namba

Experimental Cell Research 242 ( 1 ) 38 - 44 1998年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Academic Press Inc.

Transferrin (Tf), an iron-binding protein, was investigated in the cultured human fibroblast which is a major cell type in connective tissues. Tf is a major iron-transporting protein and has an important role in iron metabolism. Using two-dimensional gel electrophoresis, we demonstrated the eight subtypes of Tf produced by cultured normal human fibroblasts and their down-regulation after the immortalization of human cells, an essential early step of in vitro transformation. However, the amount of Tf mRNA in the immortalized cells was equal to that in the normal human fibroblasts, suggesting that the down-regulation occurred at the posttranscriptional level. The amount of Tf receptor increased in the immortalized cells in spite of a decrease in the amount of intracellular Tf. Interestingly the produced Tf associated with microtubules. These findings suggest a novel aspect of Tf characteristics in human fibroblasts.

DOI： 10.1006/excr.1998.4080

Two-dimensional gel electrophoretic analysis of the changes after immortalization of human cells: Decrease of intracellular alpha-2-macroglobulin fragment 査読

T Kondo, M Sakaguchi, H Yamada, M Namba

ELECTROPHORESIS 19 ( 10 ) 1836 - 1840 1998年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-V C H VERLAG GMBH

To study the mechanisms of immortalization of human cells, an early step in cancer development, we compared the cellular proteins of normal and immortalized human fibroblasts. Two-dimensional gel electrophoresis showed that one spot with a molecular mass 20 kDa and an isoelectric point of 6.0, became significantly smaller after immortalization of human cells. Further, the spot was rarely observed in four human liver cancer cell lines. Investigation of the N-terminal amino acids revealed that the spot was a fragment of alpha-2-macroglobulin. Although the 20 kDa fragment contains methionine, the spot was not labeled with [S-35]methionine. Thus we concluded that the spot might be derived from the culture medium. These results indicated that intracellular metabolism of alpha-2-macroglobulin, which is a multifunctional protease inhibitor, changed after the cells were transformed.

Differential localization of two types of transferrin: Produced by human fibroblasts or incorporated from culture medium 査読

Masakiyo Sakaguchi, Tadashi Kondo, Hong Pu, Masayoshi Namba

Cell Structure and Function 23 ( 2 ) 69 - 72 1998年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Japan Society for Cell Biology

In a previous paper, we demonstrated that cultured human fibroblasts synthesize transferrin (Tf). Two types of Tf are present
one is produced by the cells and the other is internalized from the culture medium. To study the metabolism of intracellular Tf, we investigated the subcellular localization of the two types of Tf in human fibroblasts by immunocytochemical and fluorescence-labeling techniques. The internalized Tf was found to be localized in the perinuclear area, and the synthesized Tf was associated with microtubules, forming a fibrous structure in the cytoplasm. When the cells were treated with colchicine which depolymerizes microtubules irreversibly, the synthesized Tf lost its fibrous structure and spread out in cytoplasm, but the internalized Tf remained around the nucleus. These results suggest that the two types of Tf are regulated differently in the cells.

DOI： 10.1247/csf.23.69

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