研究者詳細 - 豊國　伸哉

論文 - 豊國　伸哉

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Intraperitoneal administration of tangled multiwalled carbon nanotubes of 15 nm in diameter does not induce mesothelial carcinogenesis in rats. 査読有り

Nagai H, Okazaki Y, Chew SH, Misawa N, Miyata Y, Shinohara N and Toyokuni S.

Pathol. Int. 63 巻頁： 457-462 2013年

Metamorphosis of mesothelial cells with active horizontal motility in tissue culture. 査読有り国際誌

Nagai H, Chew SH, Okazaki Y, Funahashi S, Namba T, Kato T, Enomoto A, Jiang L, Akatsuka S, Toyokuni S

Scientific reports 3 巻頁： 1144 2013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：Scientific Reports

Mesothelial cells, which have diverse roles in physiology and pathology, constitute the mesothelium along with connective tissue and the basement membrane; the mesothelium serves to shield the somatic cavities. After mesothelial injury, mesothelial cells undergo tissue recovery. However, the mechanism of mesothelial regeneration remains poorly understood. In this study, we used confocal time-lapse microscopy to demonstrate that transformed mesothelial cells (MeT5A) and mouse peritoneal mesothelial cells can randomly migrate between cells in cell culture and in ex vivo tissue culture, respectively. Moreover, peritoneal mesothelial cells changed their morphology from a flattened shape to a cuboidal one prior to the migration. Conversely, MDCKII epithelial cells forming tight cell-cell contacts with one another do not alter the arrangement of adjacent cells during movement. Our evidence complements the current hypotheses of mesothelial regeneration and suggests that certain types of differentiated mesothelial cells undergo morphological changes before initiating migration to repair injured sites.

DOI： 10.1038/srep01144

Genotoxicity and carcinogenicity risk of carbon nanotubes. 査読有り

Toyokuni S

Adv Drug Deliver Rev 2013年

Multi-specificity of IgM antibodies raised against advanced glycation end products: Involvement of electronegative potential of antigens. 査読有り

Chikazawa M, Otaki N, Shibata T, Miyashita H, Kawai Y, Maruyama S, Toyokuni S, Kitaura Y, Matsuda T, Uchida K

J Biol Chem 288 巻頁： 13204-13214 2013年

ナノ材料（CNT）の物性と発がん性国際誌

豊國伸哉

日本毒性学会学術年会 40 巻 ( 0 ) 頁： 1044 2013年

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：日本毒性学会

1981年以降，日本人死因の第1位はがんである。喫煙や特定の感染症が発がんリスクとして同定された。しかし，産業・経済を重視するあまり，リスク評価が十分になされず，ナノマテリアルが社会に多量に持ち込まれ，がんの原因となったことも忘れてはならない。それが繊維状鉱物のアスベストであり白石綿・青石綿・茶石綿が使用された。日本では2006年に禁止となったが，アジアの諸国やロシアなどでは今も使用されている。日本の中皮腫発生ピークは2025年で今後40年間に10万人以上の方が中皮腫で死亡すると試算されている。ラットを使用して上記３種の石綿で，腹腔内10mg投与により中皮腫発がん実験を行った。2年の経過でほぼ全動物に中皮腫が発生した。石綿投与に伴い，同部の中皮細胞や貪食細胞に著明な鉄沈着を認め，Fenton反応促進性のニトリロ三酢酸の追加投与でどの石綿でも中皮腫発生が早くなった。93％の腫瘍でCdkn2A/2Bのホモ欠損を認めた。アスベスト発がんでは局所の過剰鉄病態が重要なことが示唆された。このような背景のもと，すでに中皮腫の危険性の報告のあった多層カーボンナノチューブ（CNT）の評価を行った。CNTは軽量・高強度で熱伝導性が高く導体・半導体になることからすでに電池・液晶パネルのマテリアルとして使用されているが，形状は石綿に酷似している。直径が15/50/115/150nmのCNTを使用し中皮細胞毒性実験と上記と同様のラットを使用した発がん実験を行った。中皮細胞への毒性と発がん性はほぼ一致し，50nmの発がん性が最も高かった。Cdkn2A/2Bのホモ欠損をほぼ全例で認めた。このことは，剛性が高い50nm直径のCNTは特に注意して扱うべきことを示唆している。一方，石綿はendocytosisで中皮細胞に取り込まれるが，CNTは突き刺さり入ることも明らかになった。ヒトで体腔に繊維が到達することはそう簡単ではないと考えられるが，ますます長寿化が進む現在，十分なリスク評価が必要と考えられる。  <br>

DOI： 10.14869/toxpt.40.1.0.1044.0

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脳梗塞発症前の運動による脳梗塞障害軽減効果及びその作用機序の検討〜酸化ストレスに着目して〜国際誌

野口泰司, 濱川みちる, 玉越敬悟, 戸田拓弥, 豊國伸哉, 石田和人

理学療法学Supplement 2012 巻 ( 0 ) 頁： 48101465 - 48101465 2013年

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：公益社団法人日本理学療法士協会

【はじめに、目的】脳梗塞発症時には活性酸素やフリーラジカルが大量に産生され、その酸化ストレスは組織障害を拡大させ脳梗塞を増悪させる因子である。一方で、健常なラットに長期間運動させると、脳内で抗酸化物質が増加し酸化ストレスの減少がもたらされることが報告されており、運動は脳内の抗酸化作用を高めることが示されている。また、先行研究では脳梗塞モデルラット作成前に一定期間の運動を行うと、運動していないラットに比べて、脳梗塞後の梗塞体積が減少し運動機能障害も軽減するといった脳梗塞障害軽減効果が報告されている。しかし、この運動により高まる脳内の抗酸化作用が、脳梗塞時の酸化ストレスに影響を及ぼしているかどうかは不明である。そこで本研究では、脳梗塞発症前の運動による脳梗塞障害軽減効果およびその作用機序を、脳内の抗酸化作用に着目して検討することを目的とする。【方法】実験動物にはWistar系雄性ラット（5 週齢）を用いた。無作為に（1）Sham群、（2）運動＋sham群、（3）脳梗塞群、（4）運動＋脳梗塞群に分け、脳梗塞＋運動群と運動＋sham群は3 週間のトレッドミル運動（15 m/min，30 分/日）を毎日行った。脳梗塞群とSham群は走行させずにトレッドミル装置内に暴露させた（1 日30 分間）。3 週間後、運動＋脳梗塞群と脳梗塞群に対し、小泉法による脳梗塞モデル作成手術を施行した。手術24 時間後に、運動-感覚機能評価として、麻痺の重症度を評価するneurological deficits（ND）、歩行時のバランス能力の評価としてbeam walking（BW）、はしごの上の歩行における前肢の協調運動機能の評価としてladder test、前肢の感覚運動機能の評価としてlimb placing（LP）を行った。その直後に脳を採取し、TTC染色により非梗塞半球体積に対する梗塞体積の割合を算出した。また、酸化ストレス関連指標として、脂質過酸化の指標である4-hydroxy-2-nonenal（4-HNE）とDNAの酸化的損傷の指標となる8-Hydroxydeoxyguanosine（8-OHdG）、抗酸化酵素であるthioredoxin（TRX）の免疫組織化学染色を行い、陽性細胞数及び光学濃度を計測した。またSOD Assay kit-WST（同仁化学研究所）を用いて抗酸化酵素であるsuperoxide dismutase（SOD）活性を計測した。【倫理的配慮、説明と同意】本研究における全処置は名古屋大学動物実験指針に従って実施した。【結果】運動+脳梗塞群は脳梗塞群に比べて、NDとladder testのスコアが低値となり有意に障害が軽度であった。LPとBWは群間に有意差は認められなかった。また、梗塞体積割合は運動＋脳梗塞群の方が脳梗塞群よりも有意に小さかった。4-HNE 及び8-OHdG陽性細胞数は、運動＋脳梗塞群の方が脳梗塞群に比べ有意に少なかった。一方、TRXの光学濃度は群間に有意な差は認められなかったが、SOD活性は運動＋脳梗塞群が脳梗塞群に比べ高値を示す傾向にあった。【考察】脳梗塞モデル作成前に3 週間のトレッドミル運動を継続することで、脳梗塞後の麻痺の重症度および前肢の協調運動機能障害が軽減すること、梗塞体積が縮小すること、また脳梗塞時の脂質やDNAに対する酸化ストレスを抑制することが示された。さらに抗酸化酵素であるSOD活性が高められることが示された。これらの結果より、脳梗塞前の運動による脳梗塞の障害軽減効果には酸化ストレスの抑制とSODの活性化が関与していることが示唆された。【理学療法学研究としての意義】脳梗塞発症前の運動による脳梗塞障害軽減効果を行動学的、組織化学的に示した。加えて、運動による酸化ストレスの抑制と抗酸化作用の増加の関連も示され、その作用機序の一端を明らかにした。これらの結果は、脳梗塞の予防として推奨されている運動の効果を科学的に検討し、予防医療分野における理学療法のさらなる発展に寄与するものと考える。

DOI： 10.14900/cjpt.2012.0.48101465.0

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CD146 and IMP3 predict prognosis of asbestos-induced rat mesothelioma. 査読有り

Okazaki Y, Nagai H, Chew SH, Jiang L, Funahashi S, Tsujimura T, Toyokuni S

Cnacer Sci 104 巻頁： 989-995 2013年

がんモデルマウス・ラットライブラリ　第7回　中皮腫モデル

豊國伸哉

細胞工学 32 巻頁： 220 - 224 2013年

The circadian clock gene BMAL1 is a novel therapeutic target for malignant pleural mesothelioma. 査読有り国際誌

Elshazley M, Sato M, Hase T, Yamashita R, Yoshida K, Toyokuni S, Ishiguro F, Osada H, Sekido Y, Yokoi K, Usami N, Shames DS, Kondo M, Gazdar AF, Minna JD, Hasegawa Y

International journal of cancer 131 巻 ( 12 ) 頁： 2820 - 31 2012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：International Journal of Cancer

Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm arising from the mesothelial cells lining the parietal pleura and it exhibits poor prognosis. Although there has been significant progress in MPM treatment, development of more efficient therapeutic approaches is needed. BMAL1 is a core component of the circadian clock machinery and its constitutive overexpression in MPM has been reported. Here, we demonstrate that BMAL1 may serve as a molecular target for MPM. The majority of MPM cell lines and a subset of MPM clinical specimens expressed higher levels of BMAL1 compared to a nontumorigenic mesothelial cell line (MeT-5A) and normal parietal pleural specimens, respectively. A serum shock induced a rhythmical BMAL1 expression change in MeT-5A but not in ACC-MESO-1, suggesting that the circadian rhythm pathway is deregulated in MPM cells. BMAL1 knockdown suppressed proliferation and anchorage-dependent and independent clonal growth in two MPM cell lines (ACC-MESO-1 and H290) but not in MeT-5A. Notably, BMAL1 depletion resulted in cell cycle disruption with a substantial increase in apoptotic and polyploidy cell population in association with downregulation of Wee1, cyclin B and p21WAF1/CIP1 and upregulation of cyclin E expression. BMAL1 knockdown induced mitotic catastrophe as denoted by disruption of cell cycle regulators and induction of drastic morphological changes including micronucleation and multiple nuclei in ACC-MESO-1 cells that expressed the highest level of BMAL1. Taken together, these findings indicate that BMAL1 has a critical role in MPM and could serve as an attractive therapeutic target for MPM. Copyright © 2012 UICC.

DOI： 10.1002/ijc.27598

YAP induces malignant mesothelioma cell proliferation by upregulating transcription of cell cycle-promoting genes 査読有り国際誌

Mizuno, T; Murakami, H; Fujii, M; Ishiguro, F; Tanaka, I; Kondo, Y; Akatsuka, S; Toyokuni, S; Yokoi, K; Osada, H; Sekido, Y

ONCOGENE 31 巻 ( 49 ) 頁： 5117 - 5122 2012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：Oncogene

Malignant mesothelioma (MM) shows frequent inactivation of the neurofibromatosis type 2 (NF2)-tumor-suppressor gene. Recent studies have documented that the Hippo signaling pathway, a downstream cascade of Merlin (a product of NF2), has a key role in organ size control and carcinogenesis by regulating cell proliferation and apoptosis. We previously reported that MMs show overexpression of Yes-associated protein (YAP) transcriptional coactivator, the main downstream effector of the Hippo signaling pathway, which results from the inactivation of NF2, LATS2 and/or SAV1 genes (the latter two encoding core components of the mammalian Hippo pathway) or amplification of YAP itself. However, the detailed roles of YAP remain unclear, especially the target genes of YAP that enhance MM cell growth and survival. Here, we demonstrated that YAP-knockdown inhibited cell motility, invasion and anchorage-independent growth as well as cell proliferation of MM cells in vitro. We analyzed genes commonly regulated by YAP in three MM cell lines with constitutive YAP-activation, and found that the major subsets of YAP-upregulating genes encode cell cycle regulators. Among them, YAP directly induced the transcription of CCND1 and FOXM1, in cooperation with TEAD transcription factor. We also found that knockdown of CCND1 and FOXM1 suppressed MM cell proliferation, although the inhibitory effects were less evident than those of YAP knockdown. These results indicate that constitutive YAP activation in MM cells promotes cell cycle progression giving more aggressive phenotypes to MM cells. © 2012 Macmillan Publishers Limited All rights reserved.

DOI： 10.1038/onc.2012.5

Distinct affinity of nuclear proteins to the surface of chrysotile and crocidolite. 査読有り国際誌

Kubo Y, Takenaka H, Nagai H, Toyokuni S

Journal of clinical biochemistry and nutrition 51 巻 ( 3 ) 頁： 221 - 6 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：一般社団法人　日本酸化ストレス学会

The inhalation of asbestos is a risk factor for the development of malignant mesothelioma and lung cancer. Based on the broad surface area of asbestos fibers and their ability to enter the cytoplasm and nuclei of cells, it was hypothesized that proteins that adsorb onto the fiber surface play a role in the cytotoxicity and carcinogenesis of asbestos fibers. However, little is known about which proteins adsorb onto asbestos. Previously, we systematically identified asbestos-interacting proteins and classified them into eight sub-categories: chromatin/nucleotide/RNA-binding proteins, ribosomal proteins, cytoprotective proteins, cytoskeleton-associated proteins, histones and hemoglobin. Here, we report an adsorption profile of proteins for the three commercially used asbestos compounds: chrysotile, crocidolite and amosite. We quantified the amounts of adsorbed proteins by analyzing the silver-stained gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis with ImageJ software, using the bands for amosite as a standard. We found that histones were most adsorptive to crocidolite and that chromatin-binding proteins were most adsorptive to chrysotile. The results suggest that chrysotile and crocidolite directly interact with chromatin structure through different mechanisms. Furthermore, RNA-binding proteins preferably interacted with chrysotile, suggesting that chrysotile may interfere with transcription and translation. Our results provide novel evidence demonstrating that the specific molecular interactions leading to carcinogenesis are different between chrysotile and crocidolite. ©2012 JCBN.

DOI： 10.3164/jcbn.12-39

Iron overload signature in chrysotile-induced malignant mesothelioma

Jiang L., Akatsuka S., Nagai H., Chew S.H., Ohara H., Okazaki Y., Yamashita Y., Yoshikawa Y., Yasui H., Ikuta K., Sasaki K., Kohgo Y., Hirano S., Shinohara Y., Kohyama N., Takahashi T., Toyokuni S.

Journal of Pathology 228 巻 ( 3 ) 頁： 366 - 377 2012年11月

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：Journal of Pathology

Exposure to asbestos is a risk for malignant mesothelioma (MM) in humans. Among the commercially used types of asbestos (chrysotile, crocidolite, and amosite), the carcinogenicity of chrysotile is not fully appreciated. Here, we show that all three asbestos types similarly induced MM in the rat peritoneal cavity and that chrysotile caused the earliest mesothelioma development with a high fraction of sarcomatoid histology. The pathogenesis of chrysotile-induced mesothelial carcinogenesis was closely associated with iron overload: repeated administration of an iron chelator, nitrilotriacetic acid, which promotes the Fenton reaction, significantly reduced the period required for carcinogenesis; massive iron deposition was found in the peritoneal organs with high serum ferritin; and homozygous deletion of the CDKN2A/2B/ARF tumour suppressor genes, the most frequent genomic alteration in human MM and in iron-induced rodent carcinogenesis, was observed in 92.6% of the cases studied with array-based comparative genomic hybridization. The induced rat MM cells revealed high expression of mesoderm-specific transcription factors, Dlx5 and Hand1, and showed an iron regulatory profile of active iron uptake and utilization. These data indicate that chrysotile is a strong carcinogen when exposed to mesothelia, acting through the induction of local iron overload. Therefore, an intervention to remove local excess iron might be a strategy to prevent MM after asbestos exposure. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

DOI： 10.1002/path.4075

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PubMed

Asbestos surface provides a niche for oxidative modification: A novel role of free radicals in carcinogenesis

Nagai, H; Okazaki, Y; Jiang, L; Akatsuka, S; Yamashita, Y; Toyokuni, S

FREE RADICAL BIOLOGY AND MEDICINE 53 巻頁： S51 - S52 2012年9月

Bovine lactoferrin ameliorates ferric nitrilotriacetate-induced renal oxidative damage in rats. 査読有り

Okazaki Y, Kono I, Kuriki T, Funahashi S, Fushimi S, Iqbal M, Okada S, Toyokuni S

Journal of clinical biochemistry and nutrition 51 巻 ( 2 ) 頁： 84 - 90 2012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：一般社団法人　日本酸化ストレス学会

Milk provides a well-balanced source of amino acids and other ingredients. One of the functional ingredients in milk is lactoferrin (LF). LF presents a wide variety of bioactivities and functions as a radical scavenger in models using iron-ascorbate complexes and asbestos. Human clinical trials of oral LF administration for the prevention of colon polyps have been successful and demonstrated that dietary compounds exhibit direct interactions. However, antioxidative properties of LF in distant organs require further investigation. To study the antioxidant property of LF, we employed bovine lactoferrin (bLF) using the rat model of ferric nitrilotriacetate (Fe-NTA)-induced renal tubular oxidative injury. We fed rats with bLF (0.05%, w/w) in basal chow for 4 weeks and sacrificed them after Fe-NTA treatment. After intraperitoneal administration of 9.0 mg iron/kg Fe-NTA for 4 and 24 h, bLF pretreatment suppressed elevation of serum creatinine and blood urea nitrogen levels. In addition, we observed protective effects against renal oxidative tubular damage and maintenance of antioxidant enzyme activities in the bLF-pretreated group. We thus demonstrated the antioxidative effect of bLF against Fe-NTA-induced renal oxidative injury. These results suggest that LF intake is useful for the prevention of renal tubular oxidative damage mediated by iron. ©2012 JCBN.

DOI： 10.3164/jcbn.11-100

Iron overload signature in chrysotile-induced malignant mesothelioma. 査読有り

Jiang L, Akatsuka S, Nagai H, Chew SH, Ohara H, Okazaki Y, Yamashita Y, Yoshikawa Y, Yasui H, Ikuta K, Sasaki K, Kohgo Y, Hirano S, Shinohara Y, Kohyama N, Takahashi T, Toyokuni S

J Pathol 228 巻頁： 366-377 2012年8月

Differences and similarities between carbon nanotubes and asbestos fibers during mesothelial carcinogenesis: shedding light on fiber entry mechanism. 査読有り国際誌 Open Access

Nagai H, Toyokuni S

Cancer science 103 巻 ( 8 ) 頁： 1378 - 90 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：Cancer Science

The emergence of nanotechnology represents an important milestone, as it opens the way to a broad spectrum of applications for nanomaterials in the fields of engineering, industry and medicine. One example of nanomaterials that have the potential for widespread use is carbon nanotubes, which have a tubular structure made of graphene sheets. However, there have been concerns that they may pose a potential health risk due to their similarities to asbestos, namely their high biopersistence and needle-like structure. We recently found that despite these similarities, carbon nanotubes and asbestos differ in certain aspects, such as their mechanism of entry into mesothelial cells. In the study, we showed that non-functionalized, multi-walled carbon nanotubes enter mesothelial cells by directly piercing through the cell membrane in a diameter- and rigidity-dependent manner, whereas asbestos mainly enters these cells through the process of endocytosis, which is independent of fiber diameter. In this review, we discuss the key differences, as well as similarities, between asbestos fibers and carbon nanotubes. We also summarize previous reports regarding the mechanism of carbon nanotube entry into non-phagocytic cells. As the entry of fibers into mesothelial cells is a crucial step in mesothelial carcinogenesis, we believe that a comprehensive study on the differences by which carbon nanotubes and asbestos fibers enter into non-phagocytic cells will provide important clues for the safer manufacture of carbon nanotubes through strict regulation on fiber characteristics, such as diameter, surface properties, length and rigidity. © 2012 Japanese Cancer Association.

DOI： 10.1111/j.1349-7006.2012.02326.x

Mitochondria and free radical studies on health, disease and pollution 招待有り国際誌

Majima, HJ; Toyokuni, S

FREE RADICAL RESEARCH 46 巻 ( 8 ) 頁： 925 - 6 2012年8月

The ferroimmunomodulatory role of ectopic endometriotic stromal cells in ovarian endometriosis. 査読有り国際誌

Kobayashi H, Yamashita Y, Iwase A, Yoshikawa Y, Yasui H, Kawai Y, Uchida K, Uno N, Akatsuka S, Takahashi T, Kikkawa F, Toyokuni S

Fertility and sterility 98 巻 ( 2 ) 頁： 415 - 22 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：Fertility and Sterility

Objective: To understand the role of ectopic endometriotic stromal cells in ovarian endometriosis (OEM) and the associated risks for infertility and carcinogenesis. Design: Analyses of secreted proteins and gene expression using immortalized eutopic/ectopic endometrial(-otic) stromal cells from OEM. Setting: University. Patient(s): Women with and without OEM. Intervention(s): Samples of endometrial(-otic) tissue from women with or without OEM. Main Outcome Measure(s): Immunohistochemical analysis of oxidative stress in OEM, gene expression profiles, and the identification of secreted proteins by mass spectrometry in immortalized endometrial(-otic) stromal cells. Result(s): 4-Hydroxy-2-nonenal-modified proteins and carboxymethyllysine were abundant in the stroma, rather than epithelia, of OEM patients, indicating the presence of oxidative stress. Immortalized ectopic endometriotic stromal cells exhibited high IRP1/IRP2/HIF-1β expression and contained lower amounts of iron and copper than their eutopic counterparts. Expression profiles, in combination with protein identification, revealed that complement component 3 (C3) and pentraxin-3 (PTX3) are the major proteins secreted from immortalized ectopic endometriotic stromal cells. Complement-3/PTX3 promoted the secretion of various cytokines by THP1 macrophage cells and thus supported M1 differentiation. Conclusion(s): Immortalized ectopic endometriotic stromal cells in OEM predominantly secrete C3 and PTX3 and exhibit a differential regulation of iron metabolism. Copyright © 2012 American Society for Reproductive Medicine, Published by Elsevier Inc.

DOI： 10.1016/j.fertnstert.2012.04.047

Transient but not stable ZEB1 knockdown dramatically inhibits growth of malignant pleural mesothelioma cells. 査読有り国際誌

Horio M, Sato M, Takeyama Y, Elshazley M, Yamashita R, Hase T, Yoshida K, Usami N, Yokoi K, Sekido Y, Kondo M, Toyokuni S, Gazdar AF, Minna JD, Hasegawa Y

Annals of surgical oncology 19 Suppl 3 巻 ( Suppl 3 ) 頁： S634-45 - S645 2012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：Annals of Surgical Oncology

Background: The role of ZEB1, a master epithelial-to-mesenchymal transition gene, in malignant pleural mesothelioma (MPM) is unclear. Methods: The expression of ZEB1, E-cadherin, vimentin, and epithelial cell adhesion molecule (EpCAM) in 18 MPM cell lines and a normal pleural mesothelial cell line MeT-5A was determined by quantitative real-time polymerase chain reaction and Western blot testing. RNA interference-mediated transient and/or stable knockdown of ZEB1 and EpCAM was performed. Microarray expression analysis was performed with a TORAY-3D gene chip. Growth was evaluated by colorimetric proliferation and colony formation assays. Luciferase reporter assay was performed to access the effects of ZEB1 knockdown on EpCAM promoter activity. Results: Most MPM cell lines exhibited mesenchymal phenotype and expressed ZEB1. Transient ZEB1 knockdown suppressed growth in all four cell lines studied (ACC-MESO-1, H2052, Y-MESO-8A, Y-MESO-29) while stable ZEB1 knockdown suppressed growth only in Y-MESO-29. Genome-wide gene expression analysis revealed that EpCAM was the most prominently up-regulated gene by both transient and stable ZEB1 knockdown in ACC-MESO-1, with more marked up-regulation in stable knockdown. We hypothesized that EpCAM up-regulation counteracts the stable ZEB1 knockdown-induced growth inhibition in ACC-MESO-1. Transient EpCAM knockdown suppressed growth dramatically in ACC-MESO-1 cells expressing shZEB1 but only modestly in those expressing shGFP, supporting our hypothesis. Luciferase reporter assay showed that ZEB1 knockdown resulted in increased EpCAM promoter activity. EpCAM was also up-regulated in Y-MESO-29 expressing shZEB1, but this EpCAM up-regulation did not counteract ZEB1knockdown-induced growth suppression, suggesting that the counteracting effects of EpCAM may be cellular context dependent. Conclusions: RNA interference-mediated ZEB1 knockdown may be a promising therapeutic strategy for MPM, but one has to consider the possibility of diminished growth inhibitory effects of long-term ZEB1 knockdown, possibly as a result of EpCAM up-regulation and/or other gene expression changes resulting from ZEB1 knockdown. © 2011 Society of Surgical Oncology.

DOI： 10.1245/s10434-011-2142-0

In vitro transformation of mouse testis cells by oncogene transfection. 査読有り

Morimoto H, Lee J, Tanaka T, Ishii K, Toyokuin S, Kanatsu-Shinohara M and Shinohara T.

Biol Reprod 86 巻 ( 5 ) 頁： 1-11 2012年5月

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