Papers - Imoto, Masaya

Isolation of Caldorazole, a Thiazole-Containing Polyketide with Selective Cytotoxicity under Glucose-Restricted Conditions

Osamu Ohno, Arihiro Iwasaki, Kyouhei Same, Chihiro Kudo, Erika Aida, Kazuya Sugiura, Shimpei Sumimoto, Toshiaki Teruya, Etsu Tashiro, Siro Simizu, Kenji Matsuno, Masaya Imoto, Kiyotake Suenaga

ORGANIC LETTERS (AMER CHEMICAL SOC)   2022.06

ISSN  1523-7060

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Caldorazole (1) was isolated from the marine cyanobacterium Caldora sp. collected on Ishigaki Island, Okinawa, Japan. Its structure was determined to be a new polyketide that contained two thiazole rings and an O-methylenolpyruvamide moiety. Caldorazole (1) showed strong cytotoxicity toward tumor cells that had been seeded at a high density. Cell death induced by 1 in HeLa and A431 cells was also observed only in the presence of the glycolysis blocker 2-deoxy-D-glucose (2DG). Co-treatment with 1 and 2DG remarkably decreased ATP levels in these cells. Furthermore, 1 selectively inhibited complex I in the mitochondrial respiratory chain. Thus, 1 was demonstrated to exert cytotoxicity toward human tumor cells by blocking mitochondrial respiration.

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Autophagy is Required for the Maintenance of NAD

Tetsushi Kataura, Lucia Sedlackova, Elsje G. Otten, David Shapira, Filippo Scialo, Rhoda Stefanatos, Kei-ichi Ishikawa, George Kelly, Elena Seranova, Congxin Sun, Dorothea Maetzel, Niall Kenneth, Sergey Trushin, Tong Zhang, Eugenia Trushina, Charles C. Bascom, Ryan Tasseff, Robert J. Isfort, John E. Oblong, Satomi Miwa, Michael Lazarou, Rudolf Jaenisch, Masaya Imoto, Shinji Saiki, Manolis Papamichos-Chronakis, Oliver D.K. Maddocks, Alberto Sanz, Sovan Sarkar, Viktor Korolchuk

SSRN Electronic Journal (Elsevier BV)   2022

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Involvement of miR-3180-3p and miR-4632-5p in palmitic acid-induced insulin resistance

Tashiro Etsu, Nagasawa Yumi, Itoh Susumu, Imoto Masaya

MOLECULAR AND CELLULAR ENDOCRINOLOGY 534 2021.08

ISSN  0303-7207

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Androgen receptor antagonists produced by Streptomyces overcome resistance to enzalutamide

Masaya Imoto, Takahiro Fujimaki, Shun Saito, Etsu Tashiro

The Journal of Antibiotics (Springer Science and Business Media LLC)  74 ( 10 ) 706 - 716 2021.07

ISSN  0021-8820

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Cyclopeptides from the Mushroom Pathogen Fungus Cladobotryum varium

Tao Zhou, Misaki Katsuragawa, Tian Xing, Keisuke Fukaya, Toru Okuda, Toshiyuki Tokiwa, Etsu Tashiro, Masaya Imoto, Naoya Oku, Daisuke Urabe, Yasuhiro Igarashi

JOURNAL OF NATURAL PRODUCTS (AMER CHEMICAL SOC)  84 ( 2 ) 327 - 338 2021.02

ISSN  0163-3864

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Three new cyclopeptides with serial Phe residues were identified with the aid of HPLC-DAD analysis, from the culture broth of Cladobotryum varium, a fungal pathogen causing mushroom cobweb disease. Cladoamides A (1) and B (2) have two consecutive N-methylphenylalanine units in the destruxin class cyclic depsipentapeptide framework, while cladoamide C (3) has a three consecutive Phe motif in a cyclopentapeptide structure. Of these three cyclopeptides, 1 showed potent autophagy-inducing activity at 10 mu g/mL, comparable to a positive control, rapamycin. For the determination of the absolute configurations of the Ile residues in 1 and 3, new conditions for separating Ile and alto-Ile, using a pentafluorophenyl-bonded solid phase and methanolic solvent, were established within the analytical scheme of the advanced Marfey's method, thus offering a convenient alternative to the C-3 Marfey's method, which requires elution with a threesolvent mixture. The sequence of two D-Phe and one L-Phe in 3 was determined through NMR chemical shift prediction by DFT-based calculations and chemical synthesis, which demonstrated the significance of noncovalent interactions in the accurate calculation of stable conformers for peptides with multiple aromatic rings.

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Unified synthesis and assessment of tumor cell migration inhibitory activity of optically active UTKO1, originally designed moverastin analog

Yusuke Ogura, Hiroki Kobayashi, Masaya Imoto, Hidenori Watanabe, Hirosato Takikawa

Bioscience, Biotechnology, and Biochemistry (Oxford University Press (OUP))  85 ( 1 ) 160 - 167 2021.01

ISSN  0916-8451

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<title>Abstract</title>
UTKO1 is a synthetic analog of a natural tumor cell migration inhibitor, moverastin, isolated from microbial extracts of Aspergillus sp. 7720. UTKO1 was initially developed as a mixture of the stereoisomers. In this study, a concise and unified synthesis of the 4 optically active stereoisomers of UTKO1 was achieved from a known optically pure dihydro-α-ionone through a 5-step sequence. The key transformation in the synthesis was a Nozaki–Hiyama–Kishi (NHK) reaction between an optically active enoltriflate and a known aldehyde to install the chiral allylic hydroxy group at C2′. Simple chromatographic separation of the 2 diastereomers with regard to the allylic hydroxy group was possible by the derivatization into the corresponding acetals with Nemoto's optical resolution reagent, (S)- or (R)-5-allyl-2-oxabicyclo[3.3.0]octene (ALBO). All 4 synthetic stereoisomers of UTKO1 exhibited comparable tumor cell migration inhibitory activity.

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化合物の治療有効性を評価するためのα-シヌクレインを注射したパーキンソン病(PD)モデルマウスの開発(Development of PD mouse models with alphasynuclein injection for assessment of chemical efficacy)

Ishimaru Tomohiro, Saiki Shinji, Okuzumi Ayami, Sasazawa Yukiko, Fukuhara Takeshi, Imoto Masaya, Hattori Nobutaka

臨床神経学 ((一社)日本神経学会)  60 ( Suppl. ) S502 - S502 2020.11

ISSN  0009-918X

Interaction between goniothalamin and peroxisomal multifunctional enzyme type 2 triggering endoplasmic reticulum stress

Sophonnithiprasert Thanet, Aruksakunwong Ornjira, Tashiro Etsu, Kondoh Yasumitsu, Muroi Makoto, Osada Hiroyuki, Imoto Masaya, Watanapokasin Ramida

HELIYON 6 ( 10 )  2020.10

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Miclxin, a Novel MIC60 Inhibitor, Induces Apoptosis via Mitochondrial Stress in β-Catenin Mutant Tumor Cells

Hiroaki Ikeda, Makoto Muroi, Yasumitsu Kondoh, Shumpei Ishikawa, Hideaki Kakeya, Hiroyuki Osada, Masaya Imoto

ACS Chemical Biology (American Chemical Society (ACS))  15 ( 8 ) 2195 - 2204 2020.08

ISSN  1554-8929

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A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers

Tetsushi Kataura, Etsu Tashiro, Shota Nishikawa, Kensuke Shibahara, Yoshihito Muraoka, Masahiro Miura, Shun Sakai, Naohiro Katoh, Misato Totsuka, Masafumi Onodera, Kazuo Shin-Ya, Kengo Miyamoto, Yukiko Sasazawa, Nobutaka Hattori, Shinji Saiki, Masaya Imoto

Autophagy (Informa UK Limited)  17 ( 8 ) 1 - 17 2020.08

Accepted,  ISSN  1554-8627

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Discovery of "heat shock metabolites" produced by thermotolerant actinomycetes in high-temperature culture.

Shun Saito, Wataru Kato, Hiroaki Ikeda, Yohei Katsuyama, Yasuo Ohnishi, Masaya Imoto

The Journal of antibiotics 73 ( 4 ) 203 - 210 2020.04

ISSN  0021-8820

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In actinomycetes, many secondary metabolite biosynthetic genes are not expressed under typical laboratory culture conditions and various efforts have been made to activate these dormant genes. In this study, we focused on high-temperature culture. First, we examined the thermotolerance of 3160 actinomycete strains from our laboratory culture collection and selected 57 thermotolerant actinomycetes that grew well at 45 °C. These 57 thermotolerant actinomycetes were cultured for 5 days in liquid medium at both 30 °C and 45 °C. Culture broths were extracted with 1-butanol, and each extract was subjected to LC/MS analysis. The metabolic profiles of each strain were compared between the 30 °C and 45 °C cultures. We found that almost half of these thermotolerant actinomycetes produced secondary metabolites that were detected only in the 45 °C culture. This result suggests that high-temperature culture induces the production of dormant secondary metabolites. These compounds were named "heat shock metabolites (HSMs)." To examine HSM production in more detail, 18 strains were selected at random from the initial 57 strains and cultivated in six different media at 30 °C and 45 °C; as before, metabolic profiles of each strain in each medium were compared between the 30 °C and 45 °C cultures. From this analysis, we found a total of 131 HSMs. We identified several angucycline-related compounds as HSMs from two thermotolerant Streptomyces species. Furthermore, we discovered a new compound, murecholamide, as an HSM from thermotolerant Streptomyces sp. AY2. We propose that high-temperature culture of actinomycetes is a convenient method for activating dormant secondary metabolite biosynthetic genes.

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BRUP-1, an intracellular bilirubin modulator, exerts neuroprotective activity in a cellular Parkinson's disease model.

Tetsushi Kataura, Shinji Saiki, Kei-Ichi Ishikawa, Wado Akamatsu, Yukiko Sasazawa, Nobutaka Hattori, Masaya Imoto

Journal of neurochemistry 155 ( 1 ) 81 - 97 2020.03

Accepted,  ISSN  0022-3042

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Bilirubin, the end product of heme redox metabolism, has cytoprotective properties and is an essential metabolite associated with cardiovascular disease, inflammatory bowel disease, type 2 diabetes, and neurodegenerative diseases including Parkinson's disease (PD). PD is characterized by progressive degeneration of nigral dopaminergic neurons and is associated with elevated oxidative stress due to mitochondrial dysfunction. In this study, using a ratiometric bilirubin probe, we revealed that the mitochondrial inhibitor, rotenone, which is widely used to create a PD model, significantly decreased intracellular bilirubin levels in HepG2 cells. Chemical screening showed that BRUP-1 was a top hit that restored cellular bilirubin levels that were lowered by rotenone. We found that BRUP-1 up-regulated the expression level of heme oxygenase-1 (HO-1), one of the rate-limiting enzyme of bilirubin production via nuclear factor erythroid 2-related factor 2 (Nrf2) activation. In addition, we demonstrated that this Nrf2 activation was due to a direct inhibition of the interaction between Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) by BRUP-1. Both HO-1 up-regulation and bilirubin restoration by BRUP-1 treatment were significantly abrogated by Nrf2 silencing. In neuronal PC12D cells, BRUP-1 also activated the Nrf2-HO-1 axis and increased bilirubin production, resulted in the suppression of neurotoxin-induced cell death, reactive oxygen species production, and protein aggregation, which are hallmarks of PD. Furthermore, BRUP-1 showed neuroprotective activity against rotenone-treated neurons derived from induced pluripotent stem cells. These findings provide a new member of Keap1-Nrf2 direct inhibitors and suggest that chemical modulation of heme metabolism using BRUP-1 may be beneficial for PD treatment.

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Autophagy promotes cell and organismal survival by maintaining NAD(H) pools

Lucia Sedlackova, Elsje G. Otten, Filippo Scialo, David Shapira, Tetsushi Kataura, Bernadette Carroll, Elena Seranova, Yoana Rabanal-Ruiz, George Kelly, Rhoda Stefanatos, Glyn Nelson, Francesca Urselli, Animesh Acharjee, Niall Kenneth, Sergey Trushin, Tong Zhang, Charles C. Bascom, Ryan Tasseff, Robert J. Isfort, John E. Oblong, Eugenia Trushina, Masaya Imoto, Shinji Saiki, Michael Lazarou, Manolis Papamichos Chronakis, Oliver D.K. Maddocks, Sovan Sarkar, Alberto Sanz, Viktor I. Korolchuk

(Cold Spring Harbor Laboratory)   2020.01

Accepted

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Autophagy is an essential catabolic process that promotes clearance of surplus or damaged intracellular components¹. As a recycling process, autophagy is also important for the maintenance of cellular metabolites during periods of starvation². Loss of autophagy is sufficient to cause cell death in animal models and is likely to contribute to tissue degeneration in a number of human diseases including neurodegenerative and lysosomal storage disorders^3–7. However, it remains unclear which of the many cellular functions of autophagy primarily underlies its role in cell survival. Here we have identified a critical role of autophagy in the maintenance of nicotinamide adenine dinucleotide (NAD⁺/NADH) levels. In respiring cells, loss of autophagy caused NAD(H) depletion resulting in mitochondrial membrane depolarisation and cell death. We also found that maintenance of NAD(H) is an evolutionary conserved function of autophagy from yeast to human cells. Importantly, cell death and reduced viability of autophagy-deficient animal models can be partially reversed by supplementation with an NAD(H) precursor. Our study provides a mechanistic link between autophagy and NAD(H) metabolism and suggests that boosting NAD(H) levels may be an effective intervention strategy to prevent cell death and tissue degeneration in human diseases associated with autophagy dysfunction.

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Ktedonoketone and 2’-oxosattabacin, benzenoid metabolites from a thermophilic bacterium Thermosporothrix hazakensis in the phylum Chloroflexi

Igarashi Y., Yamamoto K., Ueno C., Yamada N., Saito K., Takahashi K., Enomoto M., Kuwahara S., Oikawa T., Tashiro E., Imoto M., Xiaohanyao Y., Zhou T., Harunari E., Oku N.

Journal of Antibiotics (Journal of Antibiotics)  72 ( 9 ) 653 - 660 2019.09

Research paper (scientific journal), Joint Work, Accepted,  ISSN  00218820

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© 2019, The Author(s), under exclusive licence to the Japan Antibiotics Research Association. A thermophilic bacterium Thermosporothrix hazakensis NBRC 105916 which belongs to the class Ktedonobacteria was investigated to explore its biosynthetic potential of secondary metabolites. UV-guided fractionation led to the identification of a new benzenoid metabolite designated ktedonoketone (6) and an α-diketone metabolite 2’-oxosattabacin (7) along with five known compounds. Compound 7 was previously described as a synthetic compound, but this is the first finding as a natural product. Compound 7 induced adipocyte differentiation at 10–20 μM and autophagy at 1–10 μM. Compound 6 showed weak inducing activity of adipocyte differentiation. The biosynthetic origin of hazakacin (3), an acyloin-type compound, was elucidated by 13C-labeled precursor-feeding experiments.

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CAPZA1 determines the risk of gastric carcinogenesis by inhibiting Helicobacter pylori CagA-degraded autophagy

Tsugawa H., Mori H., Matsuzaki J., Sato A., Saito Y., Imoto M., Suematsu M., Suzuki H.

Autophagy (Autophagy)  15 ( 2 ) 242 - 258 2019.02

Research paper (scientific journal), Joint Work, Accepted,  ISSN  15548627

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© 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group. Helicobacter pylori-derived CagA, a type IV secretion system effector, plays a role as an oncogenic driver in gastric epithelial cells. However, upon delivery into gastric epithelial cells, CagA is usually degraded by macroautophagy/autophagy. Hence, the induction of autophagy in H. pylori-infected epithelial cells is an important host-protective ability against gastric carcinogenesis. However, the mechanisms by which autophagosome-lysosome fusion is regulated, are unknown. Here, we report that enhancement of LAMP1 (lysosomal associated membrane protein 1) expression is necessary for autolysosome formation. LAMP1 expression is induced by nuclear translocated LRP1 (LDL receptor related protein 1) intracellular domain (LRP1-ICD) binding to the proximal LAMP1 promoter region. Nuclear translocation of LRP1-ICD is enhanced by H. pylori infection. In contrast, CAPZA1 (capping actin protein of muscle Z-line alpha subunit 1) inhibits LAMP1 expression via binding to LRP1-ICD in the nuclei. The binding of CAPZA1 to LRP1-ICD prevents LRP1-ICD binding to the LAMP1 proximal promoter. Thus, in CAPZA1-overexpressing gastric

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Chemistry and biology for the small molecules targeting characteristics of cancer cells

Imoto M.

Bioscience, Biotechnology and Biochemistry (Bioscience, Biotechnology and Biochemistry)  83 ( 1 ) 10 - 19 2019

Research paper (scientific journal), Single Work, Accepted,  ISSN  09168451

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© 2018 Informa UK Limited, trading as Taylor & Francis Group. Despite the marked progress of cancer research, cancer is the predominant cause of death in Japan, and therefore development of effective therapeutic drugs is expected. Chemical biology is a research field utilizing small molecules to investigate biological phenomena. One of the most important aims of chemical biology is to find the small molecules, and natural products are ideal screening sources due to their structural diversity. Therefore, natural product screening based on the progress of chemical biology prompted us to find small molecules targeting cancer characteristics. Another contribution of chemical biology is to facilitate the target identification of small molecule. Therefore, among a variety of methods to uncover protein function, chemical biology is a remarkable approach in which small molecules are used as probes to elucidate protein functions related to cancer development.

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Studies of compound exerting synthetic lethality in beta-catenin mutated tumor cells

Ikeda Hiroaki, Tashiro Etsu, Imoto Masaya

CANCER SCIENCE 109   585 - 585 2018.12

Research paper (scientific journal), Accepted,  ISSN  1349-7006

Chemical genomic analysis on autophagic regulation mechanism in human lung cancer A549 cells

Kataura T, Imoto M

EUROPEAN JOURNAL OF CANCER 103   E67 - E67 2018.11

Research paper (scientific journal), Accepted,  ISSN  0959-8049

Studies of compound exerting synthetic lethality in beta-catenin mutated tumor cells

Ikeda H., Tashiro E., Imoto M.

EUROPEAN JOURNAL OF CANCER 103   E126 - E126 2018.11

Research paper (scientific journal), Accepted,  ISSN  0959-8049

14-3-3ϵa directs the pulsatile transport of basal factors toward the apical domain for lumen growth in tubulogenesis

Mizotani Y., Suzuki M., Hotta K., Watanabe H., Shiba K., Inaba K., Tashiro E., Oka K., Imoto M.

Proceedings of the National Academy of Sciences of the United States of America (Proceedings of the National Academy of Sciences of the United States of America)  115 ( 38 ) E8873 - E8881 2018.09

Research paper (scientific journal), Joint Work, Accepted,  ISSN  00278424

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© 2018 National Academy of Sciences. All rights reserved. The Ciona notochord has emerged as a simple and tractable in vivo model for tubulogenesis. Here, using a chemical genetics approach, we identified UTKO1 as a selective small molecule inhibitor of notochord tubulogenesis. We identified 14-3-3ϵa protein as a direct binding partner of UTKO1 and showed that 14-3-3ϵa knockdown leads to failure of notochord tubulogenesis. We found that UTKO1 prevents 14-3-3ϵa from interacting with ezrin/radixin/moesin (ERM), which is required for notochord tubulogenesis, suggesting that interactions between 14-3-3ϵa and ERM play a key role in regulating the early steps of tubulogenesis. Using live imaging, we found that, as lumens begin to open between neighboring cells, 14-3-3ϵa and ERM are highly colocalized at the basal cortex where they undergo cycles of accumulation and disappearance. Interestingly, the disappearance of 14-3-3ϵa and ERM during each cycle is tightly correlated with a transient flow of 14-3-3ϵa, ERM, myosin II, and other cytoplasmic elements from the basal surface toward the lumen-facing apical domain, which is often accompanied by visible changes in lumen architecture.

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Identification of protein kinase C isozymes involved in the anti-proliferative and pro-apoptotic activities of 10-Methyl-aplog-1, a simplified analog of debromoaplysiatoxin, in several cancer cell lines

Hanaki Y., Shikata Y., Kikumori M., Hotta N., Imoto M., Irie K.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications)  495 ( 1 ) 438 - 445 2018.01

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0006291X

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© 2017 Elsevier Inc. 10-Me-aplog-1 is a simplified analog of the tumor-promoting compound debromoaplysiatoxin (DAT) and a unique protein kinase C (PKC) activator with limited tumor-promoting and pro-inflammatory activities. 10-Me-aplog-1 inhibits the growth of several cancer cell lines, but the inhibitory mechanism involving PKC isozymes remains unclear. We quantified the amount of PKC isozymes in nine human cancer cell lines that differ in 10-Me-aplog-1 sensitivity. PKCα and δ were the predominant isozymes expressed in all cell lines, but there was no significant correlation between expression levels and anti-proliferative activity. Knocking down PKCα, and/or PKCδ in the three aplog-sensitive cell lines indicated their involvement in the anti-proliferative and pro-apoptotic activities of 10-Me-aplog-1. This finding suggests that PKCα and/or PKCδ activation could be effective for treating certain cancers. Since the mechanism underlying 10-Me-aplog-1's anti-proliferative activities resembles that of DAT, 10-Me-aplog-1 may be regarded as a special key derived from pleiotropic DAT as a bunch of keys.

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Professor Hamao Umezawa opened up a new road for the development of molecularly targeted therapeutic drugs for cancers

Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  71 ( 1 ) 37 - 38 2018.01

Research paper (scientific journal), Single Work, Accepted,  ISSN  00218820

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© 2018 Japan Antibiotics Research Association All rights reserved. This issue is dedicated to Prof Hamao Umezawa in appreciation of his great contributions to pioneering research on antibiotics targeting oncogenes.

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Drug discovery inspired by chemical biology

Osada Hiroyuki, Imoto Masaya

CANCER SCIENCE 109   149 - 149 2018.01

Research paper (scientific journal), Accepted,  ISSN  1349-7006

Synthetic lethal interaction of VCP inhibitor with PKA inhibitor

Tashiro Etsu, Shikata Yuki, Katoh Hiroto, Ishikawa Shumpei, Imoto Masaya

CANCER SCIENCE 109   372 - 372 2018.01

Research paper (scientific journal), Accepted,  ISSN  1349-7006

Synthetic lethal killing of beta-catenin mutant cancer cells by small molecules

Imoto Masaya

CANCER SCIENCE 109   150 - 150 2018.01

Research paper (scientific journal), Accepted,  ISSN  1349-7006

Screening of the compounds that modulate cellular bilirubin level in hepatocellular carcinoma HepG2 cells

Kataura Tetsushi, Imoto Masaya

MOLECULAR CANCER THERAPEUTICS 17 ( 1 )  2018.01

ISSN  1535-7163

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Rupture of a Calyceal Diverticulum Secondary to Ureteroscopy: A Rare Complication.

Yamasaki T, Yoshioka T, Imoto M, Aoki H, Fujio K, Uehara S, Otsuki H

Case reports in urology 2018   9285671 - 9285671 2018

Research paper (scientific journal), Accepted,  ISSN  2090-696X

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We present a case of a 45-year-old female who experienced rupture of a right calyceal diverticulum caused by ureteroscopy. Fifteen hours after the operation, she had severe right flank pain and a high fever (38.9°C). Computed tomography revealed perinephric extravasation of urine and bleeding inside the diverticulum. We diagnosed rupture of a calyceal diverticulum; therefore, we continued antibiotic administration and pain relief medication. She became afebrile on postoperative day 4 and was discharged from the hospital on postoperative day 7. Owing to renal cortex thinning in the diverticula, ureteroscopy is associated with a risk of rupture of calyceal diverticula.

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天然物スクリーニングとケミカルバイオロジーへの展開

Imoto Masaya

KAGAKU TO SEIBUTSU (Japan Society for Bioscience, Biotechnology, and Agrochemistry)  56 ( 3 ) 203 - 209 2018

Research paper (scientific journal), Accepted,  ISSN  0453-073X

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<p>天然物創薬研究が衰退しつつある今日，このたびの遠藤先生のガードナー国際賞ご受賞は「天然物スクリーニングから創薬へ」を再認識させ，これが天然物創薬研究の再活性化につながることになることを期待する．天然物スクリーニングは創薬研究だけでなく，ケミカルバイオロジー研究を展開するうえでのバイオプローブの探索でも非常に有効な手段である．ここでは，話題提供として，われわれの「創薬に至らなかった天然物スクリーニングとケミカルバイオロジーへの展開」を紹介する．</p>

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• CiNii Research

20190420

IMOTO Masaya

KAGAKU TO SEIBUTSU (Japan Society for Bioscience, Biotechnology, and Agrochemistry)  56 ( 5 ) 307 - 307 2018

Research paper (scientific journal), Accepted,  ISSN  0453-073X

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Apoptosis and autophagy

Tashiro E., Kitagawa M., Imoto M.

Bioprobes: Biochemical Tools for Investigating Cell Function: Second Edition (Bioprobes: Biochemical Tools for Investigating Cell Function: Second Edition)     75 - 113 2017.05

Research paper (scientific journal), Accepted,  ISSN  9784431565277

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© Springer Japan KK 2017. Apoptosis and autophagy are highly coordinated mechanisms to maintain cellular homeostasis against various intrinsic and/or extrinsic stresses in eukaryotic cells. Since the early 1990s, our knowledge of the molecular mechanisms and the physiological functions of apoptosis have increased substantially. Dysfunction of apoptosis leads to various diseases, including cancers and degenerative diseases. Therefore, small molecules that induce apoptosis or synthetic lethality with mutated genes have been examined for the development of cancer-specific apoptosis-inducing agents; conversely, small molecules that suppress apoptosis have been identified for protective agents against neuronal cell death. On the other hand, autophagy is an evolutionarily conserved pathway involved in the degradation of intracellular components and is critical for the maintenance of cellular homeostasis. The mechanisms and functions of autophagy have been revealed by genetic studies in yeast, which identified a series of autophagy-related genes. Many small molecules that have been discovered to induce or inhibit autophagy also provide insight into the mechanisms and functions of the autophagic process. In this chapter, we introduce several small molecules identified by synthetic lethality screening, apoptosis inhibitors, and autophagy modulators.

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Antarlides F-H, new members of the antarlide family produced by Streptomyces sp. BB47

Saito S., Fujimaki T., Panbangred W., Sawa R., Igarashi Y., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  70 ( 5 ) 595 - 600 2017.05

Research paper (scientific journal), Accepted,  ISSN  00218820

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© 2017 Japan Antibiotics Research Association All rights reserved. Castration-resistant prostate cancer (CRPC) is the most aggressive form of this disease. CRPC remains dependent on androgen receptor (AR) signaling. Therefore, a novel AR antagonist, enzalutamide, is used clinically for the treatment of men with metastatic CRPC. However, enzalutamide-resistant AR has appeared, and a new type of AR antagonist is desired. Previously, in the course of screening for a new type of AR antagonist, we isolated a series of compounds, designated antarlides A-E, that share a novel 22-membered-ring macrocyclic structure and are produced by Streptomyces sp. BB47. In the present study, we found that this strain also produces antarlides F-H as minor components. Antarlide F is a novel geometric isomer of known antarlides. On the other hand, antarlides G and H are new members of the antarlide family that have a 20-membered-ring macrocyclic structure. Antarlides G and H inhibited the binding of androgen to AR in vitro at concentrations similar to those observed with antarlides A-E. In addition, antarlide G inhibited the transcriptional activity of not only wild-type AR but also enzalutamide-resistant AR, suggesting that antarlides with either 22- or 20-membered rings may serve as potent third-generation AR antagonists capable of overcoming resistance to enzalutamide.

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Mitochondrial uncoupler exerts a synthetic lethal effect against β-catenin mutant tumor cells

Shikata Y., Kiga M., Futamura Y., Aono H., Inoue H., Kawada M., Osada H., Imoto M.

Cancer Science (Cancer Science)  108 ( 4 ) 772 - 784 2017.04

Research paper (scientific journal), Accepted,  ISSN  13479032

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© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. The wingless/int-1 (Wnt) signal transduction pathway plays a central role in cell proliferation, survival, differentiation and apoptosis. When β-catenin: a component of the Wnt pathway, is mutated into an active form, cell growth signaling is hyperactive and drives oncogenesis. As β-catenin is mutated in a wide variety of tumors, including up to 10% of all sporadic colon carcinomas and 20% of hepatocellular carcinomas, it has been considered a promising target for therapeutic interventions. Therefore, we screened an in-house natural product library for compounds that exhibited synthetic lethality towards β-catenin mutations and isolated nonactin, an antibiotic mitochondrial uncoupler, as a hit compound. Nonactin, as well as other mitochondrial uncouplers, induced apoptosis selectively in β-catenin mutated tumor cells. Significant tumor regression was observed in the β-catenin mutant HCT 116 xenograft model, but not in the β-catenin wild type A375 xenograft model, in response to daily administration of nonactin in vivo. Furthermore, we found that expression of an active mutant form of β-catenin induced a decrease in the glycolysis rate. Taken together, our results demonstrate that tumor cells with mutated β-catenin depend on mitochondrial oxidative phosphorylation for survival. Therefore, they undergo apoptosis in response to mitochondrial dysfunction following the addition of mitochondrial uncouplers, such as nonactin. These results suggest that targeting mitochondria is a potential chemotherapeutic strategy for tumor cells that harbor β-catenin mutations.

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Protein kinase A inhibition facilitates the antitumor activity of xanthohumol, a valosin-containing protein inhibitor

Shikata Y., Yoshimaru T., Komatsu M., Katoh H., Sato R., Kanagaki S., Okazaki Y., Toyokuni S., Tashiro E., Ishikawa S., Katagiri T., Imoto M.

Cancer Science (Cancer Science)  108 ( 4 ) 785 - 794 2017.04

Research paper (scientific journal), Accepted,  ISSN  13479032

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© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. Xanthohumol (XN), a simple prenylated chalcone, can be isolated from hops and has the potential to be a cancer chemopreventive agent against several human tumor cell lines. We previously identified valosin-containing protein (VCP) as a target of XN; VCP can also play crucial roles in cancer progression and prognosis. Therefore, we investigated the molecular mechanisms governing the contribution of VCP to the antitumor activity of XN. Several human tumor cell lines were treated with XN to investigate which human tumor cell lines are sensitive to XN. Several cell lines exhibited high sensitivity to XN both in vitro and in vivo. shRNA screening and bioinformatics analysis identified that the inhibition of the adenylate cyclase (AC) pathway synergistically facilitated apoptosis induced by VCP inhibition. These results suggest that there is crosstalk between the AC pathway and VCP function, and targeting both VCP and the AC pathway is a potential chemotherapeutic strategy for a subset of tumor cells.

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Metacycloprodigiosin induced cell death selectively in β-catenin-mutated tumor cells

Ikeda H., Shikata Y., Watanapokasin R., Tashiro E., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  70 ( 1 ) 109 - 112 2017.01

Research paper (scientific journal), Accepted,  ISSN  00218820

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Involvement of the MEK/ERK pathway in EGF-induced E-cadherin down-regulation

Tashiro E., Henmi S., Odake H., Ino S., Imoto M.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications)  477 ( 4 ) 801 - 806 2016.09

Research paper (scientific journal), Accepted,  ISSN  0006291X

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© 2016 Elsevier Inc. E-cadherin is a major component of the epithelial adherens junction. However, the regulatory mechanism of E-cadherin expression is still poorly understood. In this study, we found that EGF decreased E-cadherin expression at both mRNA and protein levels in colorectal carcinoma LoVo cells. Since E-cadherin down-regulation is a well-known hallmark of the EMT (Epithelial-Mesenchymal Transition), we investigated whether EGF induced E-cadherin down-regulation during the EMT. EGF was unable to affect the expression of mesenchymal markers (such as N-cadherin, vimentin or fibronectin) or EMT-regulating transcription factors (such as SNAIL, SLUG, ZEB1, ZEB2 or TWIST), suggesting that EGF induced E-cadherin down-regulation via an EMT-independent mechanism. On the other hand, the MEK inhibitor U0126 was found to suppress EGF-induced E-cadherin down-regulation at the transcriptional level, suggesting that the MEK/ERK pathway is involved in EGF-induced E-cadherin down-regulation. Moreover, we also found that EGF disrupted cell-cell contact, stimulated cells to form an elongated shape with filamentous protrusions, and induced cell migration in LoVo cells. These effects were suppressed by U0126. Therefore, EGF is suggested to induce E-cadherin down-regulation at the transcriptional level through the MEK/ERK pathway, which might result in, at least in part, the induction of cellular morphological changes and cell migration in LoVo cells.

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Antiproliferative and apoptosis induction of α-mangostin in T47D breast cancer cells

Kritsanawong S., Innajak S., Imoto M., Watanapokasin R.

International Journal of Oncology (International Journal of Oncology)  48 ( 5 ) 2155 - 2165 2016.05

Research paper (scientific journal), Accepted,  ISSN  10196439

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α-Mangostin extracted from mangosteen, Garcinia mangostana Linn. is known as 'queen of fruits'. The anticancer activity of α-mangostin through apoptosis induction and related signaling pathways in human breast cancer T47D cells was investigated. Human epidermal growth factor receptor 2 (HER2) and mitogen-activated protein kinase (MAPK) signaling have been shown to play important roles in apoptosis. The results showed that α-mangostin induced cell proliferation inhibition, DNA fragmentation, nuclear condensation, increased cleaved caspase-3 and cleaved caspase-9, but decreased Bcl-2 and Mcl-1 expression. Mitochondrial dysfunction and cytochrome c release were also detected. In addition, phosphorylation of ERα, HER2, PI3K, Akt and ERK1/2 were downregulated whereas p-JNK1/2 and p-p38 were upregulated. These results indicated that α-mangostin induced apoptosis associated with HER2/PI3K/Akt and MAPK signaling pathways suggesting that α-mangostin may be used as food supplement or a potential therapeutic compound for breast cancer.

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Ulbactins F and G, Polycyclic Thiazoline Derivatives with Tumor Cell Migration Inhibitory Activity from Brevibacillus sp.

Igarashi Y., Asano D., Sawamura M., In Y., Ishida T., Imoto M.

Organic Letters (Organic Letters)  18 ( 7 ) 1658 - 1661 2016.04

Research paper (scientific journal), Accepted,  ISSN  15237060

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© 2016 American Chemical Society. Two new structurally unique compounds bearing a nitrogen- and sulfur-containing tricyclic ring system, ulbactin F (1) and its diastereomeric isomer ulbactin G (2), were isolated from the culture extract of a sponge-derived Brevibacillus sp. The structures and absolute configurations of 1 and 2 were determined by NMR analysis and X-ray crystallographic analysis. These compounds inhibit the migration of tumor cells in the submicromolar to micromolar range.

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Chemistry and biology of the compounds that modulate cell migration

Tashiro E., Imoto M.

Journal of Industrial Microbiology and Biotechnology (Journal of Industrial Microbiology and Biotechnology)  43 ( 2-3 ) 213 - 219 2016.03

Research paper (scientific journal), Accepted,  ISSN  13675435

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© 2015, Society for Industrial Microbiology and Biotechnology. Cell migration is a fundamental step for embryonic development, wound repair, immune responses, and tumor cell invasion and metastasis. Extensive studies have attempted to reveal the molecular mechanisms behind cell migration; however, they remain largely unclear. Bioactive compounds that modulate cell migration show promise as not only extremely powerful tools for studying the mechanisms behind cell migration but also as drug seeds for chemotherapy against tumor metastasis. Therefore, we have screened cell migration inhibitors and analyzed their mechanisms for the inhibition of cell migration. In this mini-review, we introduce our chemical and biological studies of three cell migration inhibitors: moverastin, UTKO1, and BU-4664L.

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Antarlides: A New Type of Androgen Receptor (AR) Antagonist that Overcomes Resistance to AR-Targeted Therapy

Saito S., Fujimaki T., Panbangred W., Igarashi Y., Imoto M.

Angewandte Chemie - International Edition (Angewandte Chemie - International Edition)  55 ( 8 ) 2728 - 2732 2016.02

Research paper (scientific journal), Accepted,  ISSN  14337851

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© 2016 Wiley-VCH Verlag GmbH & Co. KGaA. Prostate cancer is treated with androgen receptor (AR) antagonists but most patients experience disease progression after long-term treatment with these compounds. Therefore, new AR antagonists are required for patient follow-up treatment. In the course of screening for a new AR antagonist, we isolated the novel compounds antarlides A-E (1-5) from Streptomyces sp. BB47. Antarlides are mutually isomeric with respect to the double bond and have a 22-membered-ring macrocyclic structure. The full stereostructure of 1 was established by chemical modifications, including methanolysis, the Trost method, acetonide formation, and the PGME method. 1-5 inhibited the binding of androgen to ARs in vitro. In addition, 2 inhibited the transcriptional activity of not only wild-type AR but also mutant ARs, which are seen in patients with acquired resistance to clinically used AR antagonists. Therefore, antarlides are a potent new generation of AR antagonists that overcome resistance.

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Biological activities of unique isoflavones prepared from Apios americana Medik

Kaneta H., Koda M., Saito S., Imoto M., Kawada M., Yamazaki Y., Momose I., Shindo K.

Bioscience, Biotechnology and Biochemistry (Bioscience, Biotechnology and Biochemistry)  80 ( 4 ) 774 - 778 2016

Research paper (scientific journal), Accepted,  ISSN  09168451

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© 2016 Japan Society for Bioscience, Biotechnology, and Agrochemistry. Four unique isoflavone aglycones (barpisoflavone A (1), 2′-hydroxygenistein (2), 5-methylgenistein (3), and gerontoisoflavone A (4)) whose structures were related to genistein were prepared from the tuber of Apios americana Medik. We examined the estrogen receptor and androgen receptor binding activities, estrogen agonistic activities, antioxidant activities, and α-glucosidase inhibitory activities of 1.4. The results obtained showed that 2 possessed potent and 1, 3, and 4 possessed moderate estrogen partial agonistic activities, 1 and 2 possessed moderate antioxidant activities, and 2 and 3 possessed moderate α-glucosidase inhibitory activities.

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Development of a luminescence-controllable firefly luciferin analogue using selective enzymatic cyclization

Ioka S., Saitoh T., Maki S., Imoto M., Nishiyama S.

Tetrahedron (Tetrahedron)  72 ( 47 ) 7505 - 7508 2016

Research paper (scientific journal), Accepted,  ISSN  00404020

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© 2016 Elsevier Ltd In this study, a new firefly luciferin analog that can switch firefly bioluminescence (BL) activity from ‘off’ to ‘on’ state was designed and synthesized. BL inactive N-Ac-γ-glutamate luciferin 3 contains an acyclic precursor of the thiazoline moiety. Enzymatic treatment of 3 with aminoacylase resulted in a smooth removal of the acyl protecting group and concomitant cyclization to provide BL active carboluciferin 2.

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Screening and target identification of bioactive compounds that modulate cell migration and autophagy

Tashiro E., Imoto M.

Bioorganic and Medicinal Chemistry (Bioorganic and Medicinal Chemistry)  24 ( 15 ) 3283 - 3290 2016

Research paper (scientific journal), Accepted,  ISSN  09680896

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© 2016 Elsevier Ltd Cell migration is a fundamental step for embryonic development, wound repair, immune responses, and tumor cell invasion and metastasis. It is well known that protrusive structures, namely filopodia and lamellipodia, can be observed at the leading edge of migrating cells. The formation of these structures is necessary for cell migration; however, the molecular mechanisms behind the formation of these structures remain largely unclear. Therefore, bioactive compounds that modulate protrusive structures are extremely powerful tools for studying the mechanisms behind the formation of these structures and subsequent cell migration. Therefore, we have screened for bioactive compounds that inhibit the formation of filopodia, lamellipodia, or cell migration from natural products, and attempted to identify the target molecules of our isolated compounds. Additionally, autophagy is a bulk, non-specific protein degradation system that is involved in the pathogenesis of cancer and neurodegenerative disorders. Recent extensive studies have revealed the molecular mechanisms of autophagy, however, they also remain largely unclear. Thus, we also have screened for bioactive compounds that modulate autophagy, and identified the target molecules. In the present article, we introduce the phenotypic screening system and target identification of four bioactive compounds.

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Synthesis of Firefly Luciferin Analogues and Evaluation of the Luminescent Properties

Ioka S., Saitoh T., Iwano S., Suzuki K., Maki S., Miyawaki A., Imoto M., Nishiyama S.

Chemistry - A European Journal (Chemistry - A European Journal)  22 ( 27 ) 9330 - 9337 2016

Research paper (scientific journal), Accepted,  ISSN  09476539

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© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Five new firefly luciferin (1) analogues were synthesized and their light emission properties were examined. Modifications of the thiazoline moiety in 1 were employed to produce analogues containing acyclic amino acid side chains (2–4) and heterocyclic rings derived from amino acids (5 and 6) linked to the benzothiazole moiety. Although methyl esters of all of the synthetic derivatives exhibited chemiluminescence activity, only carboluciferin (6), possessing a pyrroline-substituted benzothiazole structure, had bioluminescence (BL) activity (λmax=547 nm). Results of bioluminescence studies with AMP-carboluciferin (AMP=adenosine monophosphate) and AMP-firefly luciferin showed that the nature of the thiazoline mimicking moiety affected the adenylation step of the luciferin–luciferase reaction required for production of potent BL. In addition, BL of 6 in living mice differed from that of 1 in that its luminescence decay rate was slower.

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Chemical biology of compounds obtained from screening using disease models

Tashiro E., Imoto M.

Archives of Pharmacal Research (Archives of Pharmacal Research)  38 ( 9 ) 1651 - 1660 2015.09

Research paper (scientific journal), Accepted,  ISSN  02536269

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© 2015 The Pharmaceutical Society of Korea. Bioactive compounds are extremely powerful tools for studying biological systems because they can rapidly, conditionally, often reversibly, and dose-dependently modulate the biological function of living cells. Moreover, they are expected to be drug seeds for chemotherapy of several diseases. Two approaches are used to find and obtain bioactive compounds, namely, molecular-target-based screening and phenotypic screening. Through phenotypic screening that mimics tumor metastasis, multi-drug resistance, and Parkinson's disease, we identified several compounds that inhibit cancer cell migration, anti-apoptotic function of Bcl-2/Bcl-xL, and neuronal cell death. By using MEK inhibitor that was developed by target-based screening, we discovered that MEK inhibitor selectively induces apoptosis in tumor cells with β-catenin mutation. Using target-based screening, we identified arabilin, a novel androgen antagonist. In this review, we introduce our recent studies on the identification of bioactive compounds by phenotypic screening and by target-based screening for drug-seed discovery.

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Metabolomics and molecular targeted therapy of cancer

Tashiro E., Imoto M.

Nihon rinsho. Japanese journal of clinical medicine (Nihon rinsho. Japanese journal of clinical medicine)  73 ( 8 ) 1268 - 1272 2015.08

Research paper (scientific journal), Accepted,  ISSN  00471852

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Metabolomics is defined as the quantitative measurement of the dynamic multiparametric metabolites. Recent technological advances in the quantification of cellular metabolites, such as capillary electrophoresis (CE) -TOFMS, have prompted the comprehensive analysis of the global metabolism. Now, metabolomics has been used in the identification of new biomarker for toxicology and disease diagnosis, and the elucidation of fermentation processes. Moreover, it is considered that metabolomics would be a powerful tool for the target identification of small molecular bioactive compounds. Here we introduce our recent study that metabolomics was applied to identify the molecular target of glucopiericidin A, which was isolated through a screening of natural products for an inhibitor of cellular filopodia protrusion in carcinoma cells.

[Metabolomics and molecular targeted therapy of cancer].

Tashiro E, Imoto M

Nihon rinsho. Japanese journal of clinical medicine 73 ( 8 ) 1268 - 72 2015.08

Research paper (scientific journal), Accepted,  ISSN  0047-1852

Evaluation of drug toxicity profiles based on the phenotypes of ascidian Ciona intestinalis

Mizotani Y., Itoh S., Hotta K., Tashiro E., Oka K., Imoto M.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications)  463 ( 4 ) 656 - 660 2015.07

Research paper (scientific journal), Accepted,  ISSN  0006291X

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© 2015 Elsevier Inc. All rights reserved. In vivo toxicity evaluation using model organisms is an important step for the development of new drugs. Here, we report that Ciona intestinalis, a chordate invertebrate, is beneficial to drug toxicity evaluation for the following reasons: rapid embryonic and larval development, resemblance to vertebrates, ease of management, low cost, transparent body, and low risk of ethical issues. The dynamic phenotypic change of Ciona larvae during metamorphosis prompted us to examine the effect of cytotoxic drugs on its development by quantifying six toxicity endpoints: degenerated tail size, ampulla length, rotation of body axis, stomach size, heart rate, and body size. As a result, mitochondrial respiratory inhibitors, tubulin polymerization/depolymerization inhibitors, or DNA/RNA synthesis inhibitors showed distinct toxicity profiles against these six endpoints, but drugs with the same targets showed a similar toxicity profile in Ciona. Our results suggest Ciona is an effective animal model for profiling drug toxicity and exploring the mechanisms of drugs with unknown targets.

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Apoptosis induction associated with the ER stress response through up-regulation of JNK in HeLa cells by gambogic acid

Krajarng A., Imoto M., Tashiro E., Fujimaki T., Shinjo S., Watanapokasin R.

BMC Complementary and Alternative Medicine (BMC Complementary and Alternative Medicine)  15 ( 1 ) 26 2015.02

Research paper (scientific journal), Accepted

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© 2015 Krajarng et al. Background: Gambogic acid (GA) was extracted from the dried yellow resin of gamboge (Garcinia hanburyi) which is traditionally used as a coloring material for painting and cloth dying. Gamboge has been also used as a folk medicine for an internal purgative and externally infected wound. We focused on the mechanisms of apoptosis induction by GA through the unfold protein response (ER stress) in HeLa cells. Methods: The cytotoxic effect of GA against HeLa cells was determined by trypan blue exclusion assay. Markers of ER stress such as XBP-1, GRP78, CHOP, GADD34 and ERdj4 were analyzed by RT-PCR and Real-time RT-PCR. Cell morphological changes and apoptotic proteins were performed by Hoechst33342 staining and Western blotting technique. Results: Our results indicated a time- and dose-dependent decrease of cell viability by GA. The ER stress induction is determined by the up-regulation of spliced XBP1 mRNA and activated GRP78, CHOP, GADD34 and ERdj4 expression. GA also induced cell morphological changes such as nuclear condensation, membrane blebbing and apoptotic body in Hela cells. Apoptosis cell death detected by increased DR5, caspase-8, -9, and -3 expression as well as increased cleaved-PARP, while decreased Bcl-2 upon GA treatment. In addition, phosphorylated JNK was up-regulated but phosphorylated ERK was down-regulated after exposure to GA. Conclusions: These results suggest that GA induce apoptosis associated with the ER stress response through up-regulation of p-JNK and down-regulation of p-ERK in HeLa cells.

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SMK-17, a MEK1/2-specific inhibitor, selectively induces apoptosis in β-catenin-mutated tumors

Kiga M., Nakayama A., Shikata Y., Sasazawa Y., Murakami R., Nakanishi T., Tashiro E., Imoto M.

Scientific Reports (Scientific Reports)  5   8155 2015.02

Research paper (scientific journal), Accepted

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Although clinical studies have evaluated several MEK1/2 inhibitors, it is unlikely that MEK1/2 inhibitors will be studied clinically. BRAF mutations have been proposed as a responder marker of MEK1/2 inhibitors in a preclinical study. However, current clinical approaches focusing on BRAF mutations have shown only moderate sensitivity of MEK1/2 inhibitors. This has led to insufficient support for their promoted clinical adoption. Further characterization of tumors sensitive to MEK inhibitors holds great promise for optimizing drug therapy for patients with these tumors. Here, we report that β-catenin mutations accelerate apoptosis induced by MEK1/2 inhibitor. SMK-17, a selective MEK1/2 inhibitor, induced apoptosis in tumor cell lines harboring β-catenin mutations at its effective concentration. To confirm that β-catenin mutations and mutant β-catenin-mediated TCF7L2 (also known as TCF4) transcriptional activity is a predictive marker of MEK inhibitors, we evaluated the effects of dominant-negative TCF7L2 and of active, mutated β-catenin on apoptosis induced by MEK inhibitor. Indeed, dominant-negative TCF7L2 reduced apoptosis induced by MEK inhibitor, whereas active, mutated β-catenin accelerated it. Our findings show that β-catenin mutations are an important responder biomarker for MEK1/2 inhibitors.

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Xanthohumol suppresses oestrogen-signalling in breast cancer through the inhibition of BIG3-PHB2 interactions

Yoshimaru T., Komatsu M., Tashiro E., Imoto M., Osada H., Miyoshi Y., Honda J., Sasa M., Katagiri T.

Scientific Reports (Scientific Reports)  4   7355 2014.12

Research paper (scientific journal), Accepted

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© 2014, Nature Publishing Group. All rights reserved. Xanthohumol (XN) is a natural anticancer compound that inhibits the proliferation of oestrogen receptor-α (ERα)-positive breast cancer cells. However, the precise mechanism of the antitumour effects of XN on oestrogen (E2)-dependent cell growth, and especially its direct target molecule(s), remain(s) largely unknown. Here, we focus on whether XN directly binds to the tumour suppressor protein prohibitin 2 (PHB2), forming a novel natural antitumour compound targeting the BIG3-PHB2 complex and acting as a pivotal modulator of E2/ERα signalling in breast cancer cells. XN treatment effectively prevented the BIG3-PHB2 interaction, thereby releasing PHB2 to directly bind to both nuclear- and cytoplasmic ERα. This event led to the complete suppression of the E2-signalling pathways and ERα-positive breast cancer cell growth both in vitro and in vivo, but did not suppress the growth of normal mammary epithelial cells. Our findings suggest that XN may be a promising natural compound to suppress the growth of luminal-type breast cancer.

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Identification of licopyranocoumarin and glycyrurol from herbal medicines as neuroprotective compounds for Parkinson's disease

Fujimaki T., Saiki S., Tashiro E., Yamada D., Kitagawa M., Hattori N., Imoto M.

PLoS ONE (PLoS ONE)  9 ( 6 ) e100395 2014.06

Research paper (scientific journal), Accepted

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In the course of screening for the anti-Parkinsonian drugs from a library of traditional herbal medicines, we found that the extracts of choi-joki-to and daio-kanzo-to protected cells from MPP+-induced cell death. Because choi-joki-to and daio-kanzo-to commonly contain the genus Glycyrrhiza, we isolated licopyranocoumarin (LPC) and glycyrurol (GCR) as potent neuroprotective principals from Glycyrrhiza. LPC and GCR markedly blocked MPP +-induced neuronal PC12D cell death and disappearance of mitochondrial membrane potential, which were mediated by JNK. LPC and GCR inhibited MPP+-induced JNK activation through the suppression of reactive oxygen species (ROS) generation, thereby inhibiting MPP +-induced neuronal PC12D cell death. These results indicated that LPC and GCR derived from choi-joki-to and daio-kanzo-to would be promising drug leads for PD treatment in the future. © 2014 Fujimaki et al.

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Chemical genomic-based pathway analyses for epidermal growth factor-mediated signaling in migrating cancer cells

Magi S., Saeki Y., Kasamatsu M., Tashiro E., Imoto M.

PLoS ONE (PLoS ONE)  9 ( 5 ) e96776 2014.05

Research paper (scientific journal), Accepted

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To explore the diversity and consistency of the signaling pathways that regulate tumor cell migration, we chose three human tumor cell lines that migrated after treatment with EGF. We then quantified the effect of fifteen inhibitors on the levels of expression or the phosphorylation levels of nine proteins that were induced by EGF stimulation in each of these cell lines. Based on the data obtained in this study and chemical-biological assumptions, we deduced cell migration pathways in each tumor cell line, and then compared them. As a result, we found that both the MEK/ERK and JNK/c-Jun pathways were activated in all three migrating cell lines. Moreover, GSK-3 and p38 were found to regulate PI3K/Akt pathway in only EC109 cells, and JNK was found to crosstalk with p38 and Fos related pathway in only TT cells. Taken together, our analytical system could easily distinguish between the common and cell type-specific pathways responsible for tumor cell migration. © 2014 Magi et al.

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5-Lipoxygenase and cysteinyl leukotriene receptor 1 regulate epidermal growth factor-induced cell migration through Tiam1 upregulation and Rac1 activation

Magi S., Takemoto Y., Kobayashi H., Kasamatsu M., Akita T., Tanaka A., Takano K., Tashiro E., Igarashi Y., Imoto M.

Cancer Science (Cancer Science)  105 ( 3 ) 290 - 296 2014.03

Research paper (scientific journal), Accepted,  ISSN  13479032

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Cell migration is an essential step for tumor metastasis. The small GTPase Rac1 plays an important role in cell migration. Previously, we reported that epidermal growth factor (EGF) induced two waves of Rac1 activation; namely, at 5 min and 12 h after stimulation. A second wave of EGF-induced Rac1 activation was required for EGF-induced cell migration, however, the spatiotemporal regulation of the second wave of EGF-induced Rac1 activation remains largely unclear. In this study, we found that 5-lipoxygenase (5-LOX) is activated in the process of EGF-induced cell migration, and that leukotriene C4 (LTC4) produced by 5-LOX mediated the second wave of Rac1 activation, as well as cell migration. Furthermore, these effects caused by LTC4 were found to be blocked in the presence of the antagonist of cysteinyl leukotriene receptor 1 (CysLT1). This blockage indicates that LTC4-mediated CysLT1 signaling regulates the second EGF-induced wave of Rac1 activation. We also found that 5-LOX inhibitors, CysLT1 antagonists and the knockdown of CysLT1 inhibited EGF-induced T cell lymphoma invasion and metastasis-inducing protein 1 (Tiam1) expression. Tiam1 expression is required for the second wave of EGF-induced Rac1 activation in A431 cells. Therefore, our results indicate that the 5-LOX/LTC4/CysLT1 signaling pathway regulates EGF-induced cell migration by increasing Tiam1 expression, leading to a second wave of Rac1 activation. Thus, CysLT1 may serve as a new molecular target for antimetastatic therapy. In addition, the CysLT1 antagonist, montelukast, which is used clinically for allergy treatment, might have great potential as a novel type of antimetastatic agent. 5-lipoxygenase (5-LOX) is activated in the process of EGF-induced cell migration, and that leukotriene C4 (LTC4) produced by 5-LOX mediated Rac1 activation and cell migration. LTC4-mediated CysLT1 signaling regulates Tiam1 expression which is required for Rac1 activation. © 2013 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

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Neovessel formation promotes liver fibrosis via providing latent transforming growth factor-β

Sakata K., Eda S., Lee E., Hara M., Imoto M., Kojima S.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications)  443 ( 3 ) 950 - 956 2014.01

Research paper (scientific journal), Accepted,  ISSN  0006291X

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Aim Hepatic fibrosis and angiogenesis occur in parallel during the progression of liver disease. Fibrosis promotes angiogenesis via inducing vascular endothelial growth factor (VEGF) from the activated hepatic stellate cells (HSCs). In turn, increased neovessel formation causes fibrosis, although the underlying molecular mechanism remains undetermined. In the current study, we aimed to address a role of endothelial cells (ECs) as a source of latent transforming growth factor (TGF)-β, the precursor of the most fibrogenic cytokine TGF-β. Methods After recombinant VEGF was administered to mice via the tail vein, hepatic angiogenesis and fibrogenesis were evaluated using immunohistochemical and biochemical analyses in addition to investigation of TGF-β activation using primary cultured HSCs and liver sinusoidal ECs (LSECs). Results In addition to increased hepatic levels of CD31 expression, VEGF-treated mice showed increased α-smooth muscle actin (α-SMA) expression, hepatic contents of hydroxyproline, and latency associated protein degradation products, which reflects cell surface activation of TGF-β via plasma kallikrein (PLK). Liberating the PLK-urokinase plasminogen activator receptor complex from the HSC surface by cleaving a tethering phosphatidylinositol linker with its specific phospholipase C inhibited the activating latent TGF-β present in LSEC conditioned medium and subsequent HSC activation. Conclusion Neovessel formation (angiogenesis) accelerates liver fibrosis at least in part via provision of latent TGF-β that activated on the surface of HSCs by PLK, thereby resultant active TGF-β stimulates the activation of HSCs. © 2013 Elsevier Inc. All rights reserved.

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HCV NS3 protease enhances liver fibrosis via binding to and activating TGF-β type i receptor

Sakata K., Hara M., Terada T., Watanabe N., Takaya D., Yaguchi S., Matsumoto T., Matsuura T., Shirouzu M., Yokoyama S., Yamaguchi T., Miyazawa K., Aizaki H., Suzuki T., Wakita T., Imoto M., Kojima S.

Scientific Reports (Scientific Reports)  3   3243 2013.12

Research paper (scientific journal), Accepted

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Viruses sometimes mimic host proteins and hijack the host cell machinery. Hepatitis C virus (HCV) causes liver fibrosis, a process largely mediated by the overexpression of transforming growth factor (TGF)-β and collagen, although the precise underlying mechanism is unknown. Here, we report that HCV non-structural protein 3 (NS3) protease affects the antigenicity and bioactivity of TGF-β2 in (CAGA) 9 -Luc CCL64 cells and in human hepatic cell lines via binding to TGF-β type I receptor (TβRI). Tumor necrosis factor (TNF)-α facilitates this mechanism by increasing the colocalization of TβRI with NS3 protease on the surface of HCV-infected cells. An anti-NS3 antibody against computationally predicted binding sites for TβRI blocked the TGF-β mimetic activities of NS3 in vitro and attenuated liver fibrosis in HCV-infected chimeric mice. These data suggest that HCV NS3 protease mimics TGF-β2 and functions, at least in part, via directly binding to and activating TβRI, thereby enhancing liver fibrosis.

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Establishment of a new detection system for the dimerization of IRE1α by BiFC assay

Shinjo S., Tashiro E., Imoto M.

Bioscience, Biotechnology and Biochemistry (Bioscience, Biotechnology and Biochemistry)  77 ( 6 ) 1333 - 1336 2013.07

Research paper (scientific journal), Accepted,  ISSN  09168451

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We developed a new detection system for the activation of an endoplasmic reticulum (ER) stress sensor, inositol requiring kinase 1 α (IRE1α), by evaluating dimerization of it by bimolecular fluorescence complementation (BiFC) assay. By detecting the fluorescence derived from the reconstituted cerulean, this assay system enabled us to distinguish the activation behaviors of IRE1α as to ER stress-inducing compounds.

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Comparative analysis of the expression patterns of UPR-target genes caused by upr-inducing compounds

Shinjo S., Mizotani Y., Tashiro E., Imoto M.

Bioscience, Biotechnology and Biochemistry (Bioscience, Biotechnology and Biochemistry)  77 ( 4 ) 729 - 735 2013.06

Research paper (scientific journal), Accepted,  ISSN  09168451

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Endoplasmic reticulum (ER) stress, due to an accumulation of unfolded proteins in the ER, leads to a process known as the unfolded protein response (UPR). Since the several compounds used to induce UPR have different modes of action, their mechanisms of protein accumulation are thought to be different, but it is unclear whether these compounds can upregulate UPR target genes with similar kinetics. Hence, we sought to compare the expression patterns of nine UPR target genes induced by seven UPR-inducing compounds. Hierarchical clustering analysis revealed that the expression patterns of the UPR target genes induced by the seven compounds were classified into two clusters; cluster A (thapsigargin, tunicamycin, 2-deoxyglucose, and dithiothreitol) and cluster B (brefeldin A, monensin, and eeyarestatin I). Thus, this study suggests the existence of at least two types of UPR target gene expression profiles, which depend on the mode of action of the compounds.

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[The 14th result of screening for antineoplastic agents].

Yamori T., Uehara Y., Fukazawa H., Yoshida M., Imoto M., Kiyomiya H., Minakami T., Kawata M., Nagata H., Matsuura M.

Gan to kagaku ryoho. Cancer &amp; chemotherapy (Gan to kagaku ryoho. Cancer &amp; chemotherapy)  40 Suppl 1   18 - 114 2013.04

Research paper (scientific journal), Accepted,  ISSN  03850684

[The 15th result of screening for antineoplastic agents].

Yamori T., Uehara Y., Fukazawa H., Yoshida M., Imoto M., Kiyomiya H., Mashima T., Manakami T., Kawata M., Nagata H., Dan S., Matsuura M., Kakeya H., Tomita A.

Gan to kagaku ryoho. Cancer &amp; chemotherapy (Gan to kagaku ryoho. Cancer &amp; chemotherapy)  40 Suppl 1   116 - 147 2013.04

Research paper (scientific journal), Accepted,  ISSN  03850684

Novel isoflavone glucosides in groundnut (Apios americana medik) and their antiandrogenic activities

Ichige M., Fukuda E., Miida S., Hattan J., Misawa N., Saito S., Fujimaki T., Imoto M., Shindo K.

Journal of Agricultural and Food Chemistry (Journal of Agricultural and Food Chemistry)  61 ( 9 ) 2183 - 2187 2013.03

Research paper (scientific journal), Accepted,  ISSN  00218561

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Isoflavone glucosides (2′-hydroxy,5-methoxy genistein-7-O-glucoside (1), 2′-hydroxy genistein-7-O-gentibioside (2), 5-methoxy genistein-7-O-glucoside (3), 3′,5-dimethoxy genistein-7-O-glucoside (4), 2′-hydroxy genistein-7-O-glucoside (5), genistein-7-O-gentibioside (6), 2′-hydroxy,5-methoxy genistein-4′,7-O-diglucoside (7), and 2′-hydroxy genistein-4′,7-O-diglucoside (8)) were isolated from the groundnut of Apios americana Medik. Their structures were elucidated on the basis of HR-ESI-MS and 1D- and 2D-NMR analyses. Compounds 1, 2, 4, and 7 are new compounds presented here for the first time. Compounds 2 and 5 were proven to be androgen receptor antagonists due to their binding activities for androgen receptors (IC50 280 and 160 μM, respectively) and the inhibitory activity of androgen-induced expression of prostate-specific antigen (PSA) mRNA in LNCaP (prostate adenocarcinoma) cells (IC50 20 and 18 μM, respectively). © 2013 American Chemical Society.

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Novel derivatives of aclacinomycin A block cancer cell migration through inhibition of farnesyl transferase

Magi S., Shitara T., Takemoto Y., Sawada M., Kitagawa M., Tashiro E., Takahashi Y., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  66 ( 3 ) 165 - 170 2013.03

Research paper (scientific journal), Accepted,  ISSN  00218820

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In the course of screening for an inhibitor of farnesyl transferase (FTase), we identified two compounds, N-benzyl-aclacinomycin A (ACM) and N-allyl-ACM, which are new derivatives of ACM. N-benzyl-ACM and N-allyl-ACM inhibited FTase activity with IC 50 values of 0.86 and 2.93 μM, respectively. Not only ACM but also C-10 epimers of each ACM derivative failed to inhibit FTase. The inhibition of FTase by N-benzyl-ACM and N-allyl-ACM seems to be specific, because these two compounds did not inhibit geranylgeranyltransferase or geranylgeranyl pyrophosphate (GGPP) synthase up to 100 μM. In cultured A431 cells, N-benzyl-ACM and N-allyl-ACM also blocked both the membrane localization of H-Ras and activation of the H-Ras-dependent PI3K/Akt pathway. In addition, they inhibited epidermal growth factor (EGF)-induced migration of A431 cells. Thus, N-benzyl-ACM and N-allyl-ACM inhibited EGF-induced migration of A431 cells by inhibiting the farnesylation of H-Ras and subsequent H-Ras-dependent activation of the PI3K/Akt pathway. © 2013 Japan Antibiotics Research Association All rights reserved.

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A chemical genomic study identifying diversity in cell migration signaling in cancer cells

Magi S., Tashiro E., Imoto M.

Scientific Reports (Scientific Reports)  2   823 2012.11

Research paper (scientific journal), Accepted

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The aim of this study was to analyze the diversity and consistency of regulatory signaling in cancer cell migration, using a chemical genomic approach. The effects of 34 small molecular compounds were assessed quantitatively by wound healing assay in ten types of migrating cells. Hierarchical clustering was performed on the subsequent migration inhibition profile of the compounds and cancer cell types. The result was that hierarchical clustering accurately classified the compounds according to their targets. Furthermore, the cancer cells tested in this study were classified into three clusters, and the compounds were grouped into four clusters. An inhibitor of JNK suppressed all types of cell migration; however, inhibitors of ROCK, GSK-3 and p38MAPK only inhibited the migration of a subset of cell lines. Thus, our analytical system could easily distinguish between the common and cell type-specific signals responsible for cell migration.

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[Screening for the drug-seeds of antitumor agents].

Fujimaki T., Imoto M.

Nihon rinsho. Japanese journal of clinical medicine (Nihon rinsho. Japanese journal of clinical medicine)  70 Suppl 8   303 - 307 2012.11

Research paper (scientific journal), Accepted,  ISSN  00471852

Synthesis and assignment of the absolute configuration of indenotryptoline bisindole alkaloid BE-54017

Kimura T., Kanagaki S., Matsui Y., Imoto M., Watanabe T., Shibasaki M.

Organic Letters (Organic Letters)  14 ( 17 ) 4418 - 4421 2012.09

Research paper (scientific journal), Accepted,  ISSN  15237060

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Synthesis of the indenotryptoline bisindole alkaloid, BE-54017, was accomplished using osmium-promoted cis-dihydroxylation of maleimide as a key step. After optical resolution, the absolute configuration of this molecule was determined by comparing its optical rotation and HPLC profile to those obtained for BE-54017 derived from enantiopure cladoniamide A, whose stereochemistry has been reported previously. BE-54017 with the correct absolute stereochemistry induced apoptosis of epidermal growth factor (EGF)-stimulated EGF receptor overexpressing A431 cells and inhibited vacuolar-type H+-ATPase (V-ATPase). © 2012 American Chemical Society.

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Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing

Ri M., Tashiro E., Oikawa D., Shinjo S., Tokuda M., Yokouchi Y., Narita T., Masaki A., Ito A., Ding J., Kusumoto S., Ishida T., Komatsu H., Shiotsu Y., Ueda R., Iwawaki T., Imoto M., Iida S.

Blood Cancer Journal (Blood Cancer Journal)  2 ( 7 )  2012.07

Research paper (scientific journal), Accepted

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The IRE1a-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Therefore, the availability of small-molecule inhibitors targeting this pathway would offer a new chemotherapeutic strategy for MM. Here, we screened small-molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) activation. Furthermore, although toyocamycin was unable to inhibit IRE1α phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Toyocamycin inhibited not only ER stress-induced but also constitutive activation of XBP1 expression in MM lines as well as primary samples from patients. It showed synergistic effects with bortezomib, and induced apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM. Taken together, our results suggest toyocamycin as a lead compound for developing anti-MM therapy and XBP1 as an appropriate molecular target for anti-MM therapy. © 2012 Macmillan Publishers Limited All rights reserved.

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Comprehensive predictions of target proteins based on protein-chemical interaction using virtual screening and experimental verifications

Kobayashi H., Harada H., Nakamura M., Futamura Y., Ito A., Yoshida M., Iemura S., Shin-Ya K., Doi T., Takahashi T., Natsume T., Imoto M., Sakakibara Y.

BMC Chemical Biology (BMC Chemical Biology)  12   2 2012.06

Research paper (scientific journal), Accepted

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Background: Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis. Results: We applied our protocol of predicting target proteins combining in silico screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by in vitro binding of incednine to seven proteins, whose expression can be confirmed in our cell system.As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins. Conclusions: This study revealed that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules. © 2012 Kobayashi et al.; licensee BioMed Central Ltd.

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Inostamycin enhanced TRAIL-induced apoptosis through DR5 upregulation on the cell surface

Yamamoto K., Makino M., Watanapokasin R., Tashiro E., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  65 ( 6 ) 295 - 300 2012.06

Research paper (scientific journal), Accepted,  ISSN  00218820

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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been considered as a possible therapeutic agent for cancer treatment. This is because of its selective cytotoxicity against various cancer cells without a detrimental effect on normal cells. However, recent studies have reported that the potential application of TRAIL in cancer therapy is limited, as many cancer cells have been found to be resistant to TRAIL. Therefore, small molecule compounds that potentiate the cytotoxicity of TRAIL would be strategic candidates for therapeutic applications in combination with TRAIL. Here we found that a combined treatment of inostamycin and TRAIL synergistically induced caspase-dependent apoptosis in HCT116 cells. Inostamycin upregulated DR5, and a knockdown of DR5 suppressed the apoptosis that was synergistically induced by co-treatment with inostamycin and TRAIL. Moreover, inostamycin increased the expression of DR5 on the cell surface. Therefore, inostamycin-increased cell surface expression of DR5 may have contributed to the enhancement of TRAIL-induced apoptosis. Our study suggests that combined treatment with inostamycin and TRAIL may offer a strategy to overcome TRAIL resistance in tumor cells. © 2012 Japan Antibiotics Research Association All rights reserved.

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Xanthohumol impairs autophagosome maturation through direct inhibition of valosin-containing protein

Sasazawa Y., Kanagaki S., Tashiro E., Nogawa T., Muroi M., Kondoh Y., Osada H., Imoto M.

ACS Chemical Biology (ACS Chemical Biology)  7 ( 5 ) 892 - 900 2012.05

Research paper (scientific journal), Accepted,  ISSN  15548929

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Autophagy is a bulk, nonspecific protein degradation pathway that is involved in the pathogenesis of cancer and neurodegenerative disease. Here, we observed that xanthohumol (XN), a prenylated chalcone present in hops (Humulus lupulus L.) and beer, modulates autophagy. By using XN-immobilized beads, valosin-containing protein (VCP) was identified as a XN-binding protein. VCP has been reported to be an essential protein for autophagosome maturation. Using an in vitro pull down assay, we showed that XN bound directly to the N domain, which is known to mediate cofactor and substrate binding to VCP. These data indicated that XN inhibited the function of VCP, thereby allowing the impairment of autophagosome maturation and resulting in the accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II). This is the first report demonstrating XN as a VCP inhibitor that binds directly to the N domain of VCP. Our finding that XN bound to and inactivated VCP not only reveals the molecular mechanism of XN-modulated autophagy but may also explain how XN exhibits various biological activities that have been reported previously. © 2012 American Chemical Society.

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Napyradiomycin A1, an inhibitor of mitochondrial complexes i and II

Yamamoto K., Tashiro E., Motohashi K., Seto H., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  65 ( 4 ) 211 - 214 2012.04

Research paper (scientific journal), Accepted,  ISSN  00218820

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Target identification of bioactive compounds

Tashiro E., Imoto M.

Bioorganic and Medicinal Chemistry (Bioorganic and Medicinal Chemistry)  20 ( 6 ) 1910 - 1921 2012.03

Research paper (scientific journal), Accepted,  ISSN  09680896

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To fully understand the regulation of cellular events, functional analysis of each protein involved in the regulatory systems is required. Among a variety of methods to uncover protein function, chemical genetics is a remarkable approach in which small molecular compounds are used as probes to elucidate protein functions within signaling pathways. However, identifying the target of small molecular bioactive compounds isolated by cell-based assays represents a crucial hurdle that must be overcome before chemical genetic studies can commence. A variety of methods and technologies for identifying target proteins have been reported. This review therefore aims to describe approaches for identifying these molecular targets. © 2011 Elsevier Ltd. All rights reserved.

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Antitumor effects of novel highly hydrophilic and non-ATP-competitive MEK1/2 inhibitor, SMK-17

Kiga M., Tanzawa F., Iwasaki S., Inaba F., Fujiwara K., Iwadare H., Echigo T., Nakamura Y., Shibata T., Suzuki K., Yasumatsu I., Nakayama A., Sasazawa Y., Tashiro E., Imoto M., Kurakata S.

Anti-Cancer Drugs (Anti-Cancer Drugs)  23 ( 1 ) 119 - 130 2012.01

Research paper (scientific journal), Accepted,  ISSN  09594973

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The mitogen-activated protein kinase (MAPK) signal pathway plays a central role in regulating tumor cell proliferation, survival, and differentiation. The components of this pathway, Ras/Raf/MEK/ERK, are frequently activated in human cancers. Targeting this pathway is considered to be a promising anticancer strategy. In particular, MEK is an attractive drug target because of its high selectivity to ERK. We can expect potent growth inhibitory and proapoptotic effects by inhibiting MEK. Here, we report derivatives of N-[2-(2-chloro-4-iodo- phenylamino)-3,4-difluorophenyl]-methanesulfonamide as novel MEK1/2 inhibitors. Among these compounds, we found SMK-17 to be a potent MEK1/2 inhibitor with high aqueous solubility. The in-silico docking study suggested that SMK-17 is bound to an allosteric pocket of MEK1. The kinetic study and the kinase profiler analysis confirmed the allosteric nature of SMK-17. SMK-17 inhibited MEK1 kinase activity in a non-ATP-competitive manner and it was highly selective to MEK1 and 2. SMK-17 inhibited the growth of tumor cell lines in vitro. Especially, it seemed that cell lines harboring highly phosphorylated MEK1/2 and ERK1/2 were highly sensitive to SMK-17. Moreover, unlike previously reported MEK inhibitors, PD184352 or U0126, SMK-17 did not inhibit the phosphorylation of ERK5. In vivo, SMK-17 exhibited potent antitumor activity in animal models on oral administration. SMK-17 selectively blocked the MAPK pathway signaling without affecting other signal pathways, which resulted in significant antitumor efficacy without notable side effects. These findings suggest that SMK-17, an exquisitely selective, orally available MEK1/2 inhibitor, is a useful chemical biology tool for characterizing the function of MEK/MAPK signaling both in vitro and in vivo. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

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Involvement of 14-3-3 proteins in the second epidermal growth factor-induced wave of Rac1 activation in the process of cell migration

Kobayashi H., Ogura Y., Sawada M., Nakayama R., Takano K., Minato Y., Takemoto Y., Tashiro E., Watanabe H., Imoto M.

Journal of Biological Chemistry (Journal of Biological Chemistry)  286 ( 45 ) 39259 - 39268 2011.11

Research paper (scientific journal), Accepted,  ISSN  00219258

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Immense previous efforts have elucidated the core machinery in cell migration, actin remodeling regulated by Rho family small GTPases including RhoA, Cdc42, and Rac1; however, the spatiotemporal regulation of these molecules remains largely unknown. Here, we report that EGF induces biphasic Rac1 activation in the process of cell migration, and UTKO1, a cell migration inhibitor, inhibits the second EGF-induced wave of Rac1 activation but not the first wave. To address the regulation mechanism and role of the second wave of Rac1 activation, we identified 14-3-3ξ as a target protein of UTKO1 and also showed that UTKO1 abrogated the binding of 14-3-3ξ to Tiam1 that was responsible for the second wave of Rac1 activation, suggesting that the interaction of 14-3-3ξ with Tiam1 is involved in this event. To our knowledge, this is the first report to use a chemical genetic approach to demonstrate the mechanism of temporal activation of Rac1. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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Molecular design, chemical synthesis, and biological evaluation of agents that selectively photo-degrade the transcription factor estrogen receptor-α

Tsumura K., Suzuki A., Tsuzuki T., Tanimoto S., Kaneko H., Matsumura S., Imoto M., Umezawa K., Takahashi D., Toshima K.

Organic and Biomolecular Chemistry (Organic and Biomolecular Chemistry)  9 ( 18 ) 6357 - 6366 2011.09

Research paper (scientific journal), Accepted,  ISSN  14770520

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2-Phenylquinoline (1) degraded proteins under photo-irradiation with long-wavelength UV light without additives and under neutral conditions. We designed and synthesized a 2-phenylquinoline-estrogen receptor-α (ER-α) agonist (hybrid 2) and a 2-phenylquinoline-ER-α antagonist (hybrid 3) containing estradiol and 4-hydroxytamoxifen moieties, respectively. These 2-phenylquinoline hybrids effectively and selectively photo-degraded the target transcription factor, ER-α, which has a high affinity for estradiol and 4-hydroxytamoxifen. Target-selective photo-degradation was examined in both glass vessels and MCF-7 breast cancer cells, which are dependent upon ER-α for growth. In addition, 2-phenylquinoline-estradiol hybrid 2 functioned as an agonist of ER-α and promoted growth of MCF-7 in the absence of photo-irradiation, while it inhibited the growth of MCF-7 cells upon photo-irradiation due to the photo-degradation of ER-α. In contrast, 2-phenylquinoline-4-hydroxytamoxifen hybrid 3 inhibited growth of MCF-7 cells in the absence of photo-irradiation due to the antagonist effect of the 4-hydroxytamoxifen moiety against ER-α, and upon photo-irradiation significantly inhibited cell growth due to the dual antagonist effect of the 4-hydroxytamoxifen moiety and photo-degradation of ER-α. © 2011 The Royal Society of Chemistry.

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Generation of 'Unnatural Natural Product' library and identification of a small molecule inhibitor of XIAP

Kawamura T., Matsubara K., Otaka H., Tashiro E., Shindo K., Yanagita R., Irie K., Imoto M.

Bioorganic and Medicinal Chemistry (Bioorganic and Medicinal Chemistry)  19 ( 14 ) 4377 - 4385 2011.07

Research paper (scientific journal), Accepted,  ISSN  09680896

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Natural products have been utilized for drug discovery. To increase the source diversity, we generated a new chemical library consisting of chemically modified microbial metabolites termed 'Unnatural Natural Products' by chemical conversion of microbial metabolites in crude broth extracts followed by purification of reaction products with the LC-photo diode array-MS system. Using this library, we discovered an XIAP inhibitor, C38OX6, which restored XIAP-suppressed enzymatic activity of caspase-3 in vitro. Furthermore, C38OX6 sensitized cancer cells to anticancer drugs, whereas the unconverted natural product did not. These findings suggest that our library could be a useful source for drug seeds. © 2011 Elsevier Ltd. All rights reserved.

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Mycotrienin II, a translation inhibitor that prevents ICAM-1 expression induced by pro-inflammatory cytokines

Yamada Y., Tashiro E., Taketani S., Imoto M., Kataoka T.

Journal of Antibiotics (Journal of Antibiotics)  64 ( 5 ) 361 - 366 2011.05

Research paper (scientific journal), Accepted,  ISSN  00218820

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Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin-1α (IL-1α), trigger the activation of the transcription factor nuclear factor-B, a molecule that induces the expression of a variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Here, we report that mycotrienin II, a member of the triene-ansamycin group, inhibited the cell-surface ICAM-1 expression induced by TNF-α more strongly than that induced by IL-1α in human lung carcinoma A549 cells. Mycotrienin II was found to inhibit protein synthesis in intact living cells, as well as in cell-free translation systems. Among translation inhibitors tested, acetoxycycloheximide and anisomycin, but neither puromycin nor emetine, inhibited the TNF-α-induced ICAM-1 expression at lower concentrations than the IL-1α-induced ICAM-1 expression. Several compounds of the triene-ansamycin group (that is, mycotrienin I, trienomycin A, trierixin, quinotrierixin and quinotrierixin HQ) also inhibited ICAM-1 expression, as well as cell-free translation in a manner similar to mycotrienin II. These results indicate that mycotrienin II is a direct inhibitor of translation, thereby inhibiting ICAM-1 expression induced by pro-inflammatory cytokines. © 2011 Japan Antibiotics Research Association. All rights reserved.

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A new, convenient cell-based screening method for small-molecule glycolytic inhibitors

Kitagawa M., Misawa M., Ogawa S., Tashiro E., Imoto M.

Bioscience, Biotechnology and Biochemistry (Bioscience, Biotechnology and Biochemistry)  75 ( 2 ) 367 - 369 2011.03

Research paper (scientific journal), Accepted,  ISSN  09168451

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To counteract active glycolysis in tumors, we developed a new, convenient cell-based screening system to identify an inhibitor of glycolysis. Using this system, we searched for an inhibitor in the synthetic Carbasugar library and found two candidates. It was found that both inhibited glycolysis by suppressing the glucose uptake step in tumor cells.

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Quinotrierixin inhibited ER stress-induced XBPl mRNA splicing through inhibition of protein synthesis

Yamamoto K., Tashiro E., Imoto M.

Bioscience, Biotechnology and Biochemistry (Bioscience, Biotechnology and Biochemistry)  75 ( 2 ) 284 - 288 2011.03

Research paper (scientific journal), Accepted,  ISSN  09168451

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Quinotrierixin was isolated from microbes as an inhibitor of ER stress-induced XBPl mRNA splicing, but its mode of action was unclear. We found that quinotrierixin is an inhibitor of protein synthesis, and that the required dose range of quinotrierixin to inhibit ER stress-induced XBPl mRNA splicing was similar to that to inhibit protein synthesis. Furthermore, we also found that quinotrierixin inhibited the ER stress-induced increases of unfolded protein response-related genes such as GRP78, CHOP, EDEM, ERdj4, and p58IPK. Thus, we showed that quinotrierixin inhibited the ER stress-induced unfolded protein response, possibly due to its inhibitory activity of protein synthesis.

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Synthesis and anti-migrative evaluation of moverastin derivatives

Sawada M., Kubo S., Matsumura K., Takemoto Y., Kobayashi H., Tashiro E., Kitahara T., Watanabe H., Imoto M.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  21 ( 5 ) 1385 - 1389 2011.03

Research paper (scientific journal), Accepted,  ISSN  0960894X

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Cell migration of tumor cells is essential for invasion of the extracellular matrix and for cell dissemination. Inhibition of the cell migration involved in the invasion process represents a potential therapeutic approach to the treatment of tumor metastasis; therefore, a novel series of derivatives of moverastins (moverastins A and B), an inhibitor of tumor cell migration, was designed and chemically synthesized. Among these moverastin derivatives, several compounds showed stronger cell migration inhibitory activity than parental moverastins, and UTKO1 was found to have the most potent inhibitory activity against the migration of human esophageal tumor EC17 cells in a chemotaxis cell chamber assay. Interestingly, although moverastins are considered to inhibit tumor cell migration by inhibiting farnesyltransferase (FTase), UTKO1 did not inhibit FTase, indicating that UTKO1 inhibited tumor cell migration by a mechanism other than the inhibition of FTase. © 2011 Published by Elsevier Ltd.

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Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition

Saiki S., Sasazawa Y., Imamichi Y., Kawajiri S., Fujimaki T., Tanida I., Kobayashi H., Sato F., Sato S., Ishikawa K., Imoto M., Hattori N.

Autophagy (Autophagy)  7 ( 2 ) 176 - 187 2011.02

Research paper (scientific journal), Accepted,  ISSN  15548627

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Caffeine is one of the most frequently ingested neuroactive compounds. All known mechanisms of apoptosis induced by caffeine act through cell cycle modulation or p53 induction. It is currently unknown whether caffeine-induced apoptosis is associated with other cell death mechanisms, such as autophagy. Herein we show that caffeine increases both the levels of microtubule-associated protein 1 light chain 3-II and the number of autophagosomes, through the use of western blotting, electron microscopy and immunocytochemistry techniques. Phosphorylated p70 ribosomal protein S6 kinase (Thr389), S6 ribosomal protein (Ser235/236), 4E-BP1 (Thr37/46) and Akt (Ser473) were significantly decreased by caffeine. In contrast, ERK1/2 (Thr202/204) was increased by caffeine, suggesting an inhibition of the Akt/mTOR/p70S6K pathway and activation of the ERK1/2 pathway. Although insulin treatment phosphorylated Akt (Ser473) and led to autophagy suppression, the effect of insulin treatment was completely abolished by caffeine addition. Caffeine-induced autophagy was not completely blocked by inhibition of ERK1/2 by U0126. Caffeine induced reduction of mitochondrial membrane potentials and apoptosis in a dose-dependent manner, which was further attenuated by the inhibition of autophagy with 3-methyladenine or Atg7 siRNA knockdown. Furthermore, there was a reduced number of early apoptotic cells (annexin V positive, propidium iodide negative) among autophagy-deficient mouse embryonic fibroblasts treated with caffeine than in their wild-type counterparts. These results support previous studies on the use of caffeine in the treatment of human tumors and indicate a potential new target in the regulation of apoptosis. © 2011 Landes Bioscience.

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Total synthesis of incednam, the aglycon of incednine

Ohtani T., Tsukamoto S., Kanda H., Misawa K., Urakawa Y., Fujimaki T., Imoto M., Takahashi Y., Takahashi D., Toshima K.

Organic Letters (Organic Letters)  12 ( 21 ) 5068 - 5071 2010.11

Research paper (scientific journal), Accepted,  ISSN  15237060

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The first total synthesis of incednam (1), the aglycon of antibiotic incednine (2), is described. Incednine has been reported to exhibit significant inhibitory activity against the antiapoptotic oncoproteins Bcl-2 and Bcl-xL. The synthesis of 1 commenced with the preparation of the C1-C13 subunit 3 and the C14-C23 subunit 4. The construction of the novel 24-membered macrocycle was achieved by the application of a Stille coupling between 3 and 4, followed by macrolactamization. © 2010 American Chemical Society.

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Isolation and structure elucidation of a novel androgen antagonist, arabilin, produced by Streptomyces sp. MK756-CF1

Kawamura T., Fujimaki T., Hamanaka N., Torii K., Kobayashi H., Takahashi Y., Igarashi M., Kinoshita N., Nishimura Y., Tashiro E., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  63 ( 10 ) 601 - 605 2010.10

Research paper (scientific journal), Accepted,  ISSN  00218820

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In the course of screening for a new type of androgen receptor (AR) antagonist, we isolated a novel compound, arabilin, with two structural isomers, spectinabilin and SNF4435C, produced by Streptomyces sp. MK756-CF1. Structure elucidation on the basis of the spectroscopic properties showed that arabilin is a novel polypropionate-derived metabolite with a p-nitrophenyl group and a substituted γ-pyrone ring. Arabilin competitively blocked the binding of androgen to the ligand-binding domain of AR in vitro. In addition, arabilin inhibited androgen-induced prostate-specific antigen mRNA expression in prostate cancer LNCaP cells. © 2010 Japan Antibiotics Research Association All rights reserved.

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Metabolomic identification of the target of the filopodia protrusion inhibitor glucopiericidin A

Kitagawa M., Ikeda S., Tashiro E., Soga T., Imoto M.

Chemistry and Biology (Chemistry and Biology)  17 ( 9 ) 989 - 998 2010.09

Research paper (scientific journal), Accepted,  ISSN  10745521

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Identifying the targets of bioactive compounds is a major challenge in chemical biological research. Here, we identified the functional target of the natural bioactive compound glucopiericidin A (GPA) through metabolomic analysis. We isolated GPA while screening microbial samples for a filopodia protrusion inhibitor. Interestingly, GPA alone did not inhibit filopodia protrusion, but synergistically inhibit protrusion with the mitochondrial respiration inhibitor, piericidin A (PA). These results suggested that GPA might inhibit glycolysis. Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) provided strong evidence that GPA suppresses glycolysis by functionally targeting the glucose transporter. GPA may therefore serve as a glucose transporter chemical probe. Simultaneous inhibition of both glycolysis and mitochondrial respiration dramatically decreased intracellular ATP levels, indicating that GPA inhibits ATP-dependent filopodia protrusion with PA. Our results represent a challenge of molecular target identification using metabolomic analysis. © 2010 Elsevier Ltd. All rights reserved.

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Glyoxalase-I is a novel target against Bcr-Abl leukemic cells acquiring stem-like characteristics in a hypoxic environment

Takeuchi M., Kimura S., Kuroda J., Ashihara E., Kawatani M., Osada H., Umezawa K., Yasui E., Imoto M., Tsuruo T., Yokota A., Tanaka R., Nagao R., Nakahata T., Fujiyama Y., Maekawa T.

Cell Death and Differentiation (Cell Death and Differentiation)  17 ( 7 ) 1211 - 1220 2010.07

Research paper (scientific journal), Accepted,  ISSN  13509047

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Abl tyrosine kinase inhibitors (TKIs) such as imatinib and dasatinib are ineffective against Bcr-Abl leukemic stem cells. Thus, the identification of novel agents that are effective in eradicating quiescent Bcr-Abl stem cells is needed to cure leukemias caused by Bcr-Abl cells. Human Bcr-Abl cells engrafted in the bone marrow of immunodeficient mice survive under severe hypoxia. We generated two hypoxia-adapted (HA)-Bcr-Abl sublines by selection in long-term hypoxic cultures (1.0% O 2). Interestingly, HA-Bcr-Abl cells exhibited stem cell-like characteristics, including more cells in a dormant, increase of side population fraction, higher Β-catenin expression, resistance to Abl TKIs, and a higher transplantation efficiency. Compared with the respective parental cells, HA-Bcr-Abl cells had higher levels of protein and higher enzyme activity of glyoxalase-I (Glo-I), an enzyme that detoxifies methylglyoxal, a cytotoxic by-product of glycolysis. In contrast to Abl TKIs, Glo-I inhibitors were much more effective in killing HA-Bcr-Abl cells both in vitro and in vivo. These findings indicate that Glo-I is a novel molecular target for treatment of Bcr-Abl leukemias, and, in particular, Abl TKI-resistant quiescent Bcr-Abl leukemic cells that have acquired stem-like characteristics in the process of adapting to a hypoxic environment. © 2010 Macmillan Publishers Limited All rights reserved.

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Terpenoids produced by actinomycetes: Isolation, structural elucidation and biosynthesis of new diterpenes, gifhornenolones A and B from Verrucosispora gifhornensis YM28-088

Shirai M., Okuda M., Motohashi K., Imoto M., Furihata K., Matsuo Y., Katsuta A., Shizuri Y., Seto H.

Journal of Antibiotics (Journal of Antibiotics)  63 ( 5 ) 245 - 250 2010.05

Research paper (scientific journal), Accepted,  ISSN  00218820

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New terpenoids named gifhornenolones A (1) and B (2) were isolated from the culture broth of Verrucosispora gifhornensis YM28-088, and their structures were established as hydroxylated isopimaradiene derivatives on the basis of extensive NMR and MS spectral analyses. In addition, a known sesquiterpene compound cyperusol C (3) was isolated. The absolute configuration of 1 was determined by nuclear Overhauser effect spectroscopy (NOESY) and CD spectra as 4R, 5S, 9R, 10S, 13R, and that of 2 was determined by NOESY experiments as 3R, 4R, 5R, 9R, 10S, 13R. Labeling experiments with 1-13 Cglucose and U-13 C6 glucose confirmed that the MEP (2-C-methyl-D-erythritol-4-phosphate) pathway was used for the biosynthesis of terpenoids in this organism. 1 showed potent inhibitory activity to the androgen receptor with an IC50 of 2.8 μg ml-1. © 2010 Japan Antibiotics Research Association All rights reserved.

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Distinct subcellular localization in the cytosol and apicoplast, unexpected dimerization and inhibition of Plasmodium falciparum glyoxalases

Urscher M., Przyborski J., Imoto M., Deponte M.

Molecular Microbiology (Molecular Microbiology)  76 ( 1 ) 92 - 103 2010.04

Research paper (scientific journal), Accepted,  ISSN  0950382X

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The ubiquitous glyoxalase system removes methylglyoxal as a harmful by-product of glycolysis. Because malaria parasites have drastically increased glycolytic fluxes, they could be highly susceptible to the inhibition of this detoxification pathway. Here we analysed the intracellular localization, oligomerization and inhibition of the glyoxalases from Plasmodium falciparum. Glyoxalase I (GloI) and one of the two glyoxalases II (cGloII) were located in the cytosol of the blood stages. The second glyoxalase II (tGloII) was detected in the apicoplast pointing to alternative metabolic pathways. Using a variety of methods, cGloII was found to exist in a monomer-dimer equilibrium that might have been overlooked for homologues from other organisms and that could be of physiological importance. The compounds methyl-gerfelin and curcumin, which were previously shown to inhibit mammalian GloI, also inhibited P. falciparum GloI. Inhibition patterns were predominantly competitive but were complicated because of the two different active sites of the enzyme. This effect was neglected in previous inhibition studies of monomeric glyoxalases I, with consequences for the interpretation of inhibition constants. In summary, the present work reveals novel general glyoxalase properties that future research can build on and provides a significant advance in characterizing the glyoxalase system from P. falciparum. © 2010 Blackwell Publishing Ltd.

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Mesenchyme cells can function to induce epithelial cell proliferation in starfish embryos

Hamanaka G., Matsumoto M., Imoto M., Kaneko H.

Developmental Dynamics (Developmental Dynamics)  239 ( 3 ) 818 - 827 2010.03

Research paper (scientific journal), Accepted,  ISSN  10588388

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Here, we show that mesenchyme cells have a novel morphogenetic function in epithelial cell proliferation in starfish embryos. Blastula embryos were injected with pure populations of mesenchyme cells and the total cell numbers in the treated embryos were subsequently determined at different developmental stages. When a total of 40-50 mesenchyme cells was injected, total cells numbers in mid-gastrula embryos and 3-day-old bipinnaria larvae increased significantly (by 1.3-fold) compared with controls, with no indication of any mitotic activity in the injected mesenchyme cells. However, injection of more than 150 mesenchyme cells failed to induce proliferation of the epithelial cells and, moreover, interfered with normal morphogenesis. These developmental abnormalities occurred concomitantly with a severe condensation of the fibrous component of the extracellular matrix. Our data suggest that epithelial cell proliferation is induced by an appropriate number of mesenchyme cells in concert with the fibrous component of the extracellular matrix. © 2010 Wiley-Liss, Inc.

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Chemical Biology of Cell Motility Inhibitors

Kawamura T., Kitagawa M., Imoto M.

Protein Targeting with Small Molecules: Chemical Biology Techniques and Applications (Protein Targeting with Small Molecules: Chemical Biology Techniques and Applications)     169 - 187 2009.08

Research paper (scientific journal), Accepted,  ISSN  9780470120538

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Oligomycin induced the proteasomal degradation of cyclin D1 protein

Kanai M., Iba S., Okada R., Tashiro E., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  62 ( 8 ) 425 - 429 2009.08

Research paper (scientific journal), Accepted,  ISSN  00218820

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We searched for compounds that affect the cyclin D1/retinoblastoma protein pathway from the in-house natural product library using a recombinant adenovirus with the Cre/loxP-regulated cyclin D1 overexpression system, and we found that oligomycin inhibited cell growth more effectively in cyclin D1-overexpressing SW480 cells than in control SW480 cells. We also found that oligomycin reduced the expression levels of cyclin D1 protein and that this reduction is, at least in part, mediated by Thr-286 phosphorylation-dependent proteasomal degradation. © 2009 Japan Antibiotics Research Association.

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Vacuolar H⁺-ATPase inhibitors overcome Bcl-xL-mediated chemoresistance through restoration of a caspase-independent apoptotic pathway

Sasazawa Y., Futamura Y., Tashiro E., Imoto M.

Cancer Science (Cancer Science)  100 ( 8 ) 1460 - 1467 2009.07

Research paper (scientific journal), Accepted,  ISSN  13479032

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The anti-apoptotic oncoproteins Bcl-2 and Bcl-xL play crucial roles in tumorigenesis and chemoresistance, and are thus therapeutic cancer targets. We searched for small molecules that disturbed the anti-apoptotic function of Bcl-2 or Bcl-xL, and found vacuolar H+-ATPase (V-ATPase) inhibitors, such as bafilomycin A1 (BMA), that showed such activity. Bcl-xL-overexpressing Ms-1 cells displayed resistance to anticancer drugs, but underwent apoptosis following treatment with a combination of V-ATPase inhibitors at doses similar to those that caused inhibitory activities of V-ATPase. We investigated the apoptosis mechanism induced by cotreatment of Bcl-xL-overexpressing Ms-1 cells with BMA as a V-ATPase inhibitor and taxol (TXL) as an anticancer drug. With BMA, TXL triggered mitochondrial membrane potential loss and cytochrome c release, whereas downstream caspase activation was not observed. In contrast, pronounced nuclear translocation of mitochondrial apoptosis-inducing factor and endonuclease G, known as effectors of caspase-independent apoptosis, was observed with BMA and TXL cotreatment. Moreover, depletion of apoptosis-inducing factor and endonuclease G using each siRNA significantly rescued cells from BMA- and TXL-induced apoptosis. Hence, the apoptosis-inducing factor- and endonuclease G-dependent pathway was critical for apoptosis induction by BMA and TXL cotreatment. Our data suggest that V-ATPase inhibitors could not only suppress anti-apoptotic Bcl-2 nor Bcl-xL but could also facilitate the caspase-independent apoptotic pathway. V-ATPase inhibition will be a promising therapeutic approach for Bcl-2- or Bcl-xL-overexpressing malignancies. © 2009 Japanese Cancer Association.

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Transcriptional regulation of human fibroblast growth factor receptor 1 by E2F-1

Kanai M., Tashiro E., Maruki H., Minato Y., Imoto M.

Gene (Gene)  438 ( 1-2 ) 49 - 56 2009.06

Research paper (scientific journal), Accepted,  ISSN  03781119

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Overexpression of fibroblast growth factor receptors (FGFRs) has been observed in many types of human tumors; however, the regulatory mechanism of human FGFR expression is still largely unknown. In the present study, we first identified the transcriptional initiation site in the human FGFR 1 gene by 5′-RACE. Furthermore, we show that the expression of human FGFR 1 is regulated by E2F-1. Characterization of the human FGFR 1 promoter demonstrated that two non-consensus E2F binding sequences at positions + 4 to + 22 and + 25 to + 43 relative to our identified transcriptional initiation site in the human FGFR 1 gene were critical for E2F-1-mediated transactivation of human FGFR 1 promoter. Mutations of these sites completely abolished the response of human FGFR 1 promoter to E2F-1 as well as E2F-1 binding in electrophoretic mobility-shift assays. Furthermore, chromatin immunoprecipitation assay showed that E2F-1 was able to bind in vivo to the human FGFR 1 promoter. Moreover, human FGFR 1 protein expression was up-regulated by the overexpression of E2F-1, but down-regulated by the overexpression of pRB in situ, suggesting that the expression of human FGFR 1 is regulated by the pRB/E2F pathway. Because disruption of the pRB/E2F pathway is frequently observed in tumor cells, our findings provide valuable information for studying the role of FGFR 1 in tumor progression. © 2009 Elsevier B.V. All rights reserved.

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Integrating statistical predictions and experimental verifications for enhancing protein-chemical interaction predictions in virtual screening

Nagamine N., Shirakawa T., Minato Y., Torii K., Kobayashi H., Imoto M., Sakakibara Y.

PLoS Computational Biology (PLoS Computational Biology)  5 ( 6 ) e1000397 2009.06

Research paper (scientific journal), Accepted,  ISSN  1553734X

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Predictions of interactions between target proteins and potential leads are of great benefit in the drug discovery process. We present a comprehensively applicable statistical prediction method for interactions between any proteins and chemical compounds, which requires only protein sequence data and chemical structure data and utilizes the statistical learning method of support vector machines. In order to realize reasonable comprehensive predictions which can involve many false positives, we propose two approaches for reduction of false positives: (i) efficient use of multiple statistical prediction models in the framework of two-layer SVM and (ii) reasonable design of the negative data to construct statistical prediction models. In two-layer SVM, outputs produced by the first-layer SVM models, which are constructed with different negative samples and reflect different aspects of classifications, are utilized as inputs to the second-layer SVM. In order to design negative data which produce fewer false positive predictions, we iteratively construct SVM models or classification boundaries from positive and tentative negative samples and select additional negative sample candidates according to pre-determined rules. Moreover, in order to fully utilize the advantages of statistical learning methods, we propose a strategy to effectively feedback experimental results to computational predictions with consideration of biological effects of interest. We show the usefulness of our approach in predicting potential ligands binding to human androgen receptors from more than 19 million chemical compounds and verifying these predictions by in vitro binding. Moreover, we utilize this experimental validation as feedback to enhance subsequent computational predictions, and experimentally validate these predictions again. This efficient procedure of the iteration of the in silico prediction and in vitro or in vivo experimental verifications with the sufficient feedback enabled us to identify novel ligand candidates which were distant from known ligands in the chemical space. © 2009 Nagamine et al.

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Bioprobe generation from microbial origin for chemical biology study

Imoto M.

Folia Pharmacologica Japonica (Folia Pharmacologica Japonica)  132 ( 1 ) 26 - 30 2008.09

Research paper (scientific journal), Accepted,  ISSN  00155691

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The identification of an osteoclastogenesis inhibitor through the inhibition of glyoxalase I

Kawatani M., Okumura H., Honda K., Kanoh N., Muroi M., Dohmae N., Takami M., Kitagawa M., Futamura Y., Imoto M., Osada H.

Proceedings of the National Academy of Sciences of the United States of America (Proceedings of the National Academy of Sciences of the United States of America)  105 ( 33 ) 11691 - 11696 2008.08

Research paper (scientific journal), Accepted,  ISSN  00278424

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Osteoclasts, bone-resorptive multinucleated cells derived from hematopoietic stem cells, are associated with many bone-related diseases, such as osteoporosis. Osteoclast-targeting small-molecule inhibitors are valuable tools for studying osteoclast biology and for developing antiresorptive agents. Here, we have discovered that methyl-gerfelin (M-GFN), the methyl ester of the natural product gerfelin, suppresses osteoclastogenesis. By using M-GFN-immobilized beads, glyoxalase I (GLO1) was identified as an M-GFN-binding protein. GLO1 knockdown and treatment with an established GLO1 inhibitor in osteoclast progenitor cells interfered with osteoclast generation, suggesting that GLO1 activity is required for osteoclastogenesis. In cells, GLO1 plays a critical role in the detoxification of 2-oxoaldehydes, such as methylglyoxal. M-GFN inhibited the enzymatic activity of GLO1 in vitro and in situ. Furthermore, the cocrystal structure of the GLO1/M-GFN complex revealed the binding mode of M-GFN at the active site of GLO1. These results suggest that M-GFN targets GLO1, resulting in the inhibition of osteoclastogenesis. © 2008 by The National Academy of Sciences of the USA.

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Hot springs and cool natural products

Kwon H., Lee C., Osada H., Yoshida M., Imoto M.

Nature Chemical Biology (Nature Chemical Biology)  4 ( 8 ) 444 - 446 2008.08

Research paper (scientific journal), Accepted,  ISSN  15524450

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SAR study of a novel triene-ansamycin group compound, quinotrierixin, and related compounds, as inhibitors of ER stress-induced XBP1 activation I. Taxonomy, fermentation, isolation, biological activities and SAR study

Kawamura T., Tashiro E., Yamamoto K., Shindo K., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  61 ( 5 ) 303 - 311 2008.05

Research paper (scientific journal), Accepted,  ISSN  00218820

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In the course of screening for an inhibitor of ER stress-induced XBP1 activation, we isolated a new member of the triene-ansamycin group compound, quinotrierixin, from a culture broth of Streptomyces sp. PAE37. Quinotrierixin inhibited thapsigargin-induced XBP1 activation in HeLa cells with an IC 50 of 0.067 μM. We found that other triene-ansamycin group compounds such as demethyltrienomycin A and mycotrienin I also inhibited ER stress-induced XBP1 activation. Moreover, we performed SAR study of twelve triene-ansamycin group compounds. The study showed that OH group at C-13 was crucial, and CH3 group at C-14 would be important for the XBP1 inhibitory activity. © Japan Antibiotics Research Association.

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SAR study of a novel triene-ansamycin group compound, quinotrierixin, and related compounds, as inhibitors of ER stress-induced XBP1 activation.

Kawamura T, Tashiro E, Shindo K, Imoto M

The Journal of antibiotics 61 ( 5 ) 312 - 7 2008.05

Research paper (scientific journal), Accepted,  ISSN  0021-8820

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SAR study of a novel triene-ansamycin group compound, quinotrierixin, and related compounds, as inhibitors of ER stress-induced XBP1 activation.

Kawamura T, Tashiro E, Yamamoto K, Shindo K, Imoto M

The Journal of antibiotics 61 ( 5 ) 303 - 11 2008.05

Research paper (scientific journal), Accepted,  ISSN  0021-8820

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SAR study of a novel triene-ansamycin group compound, quinotrierixin, and related compounds, as inhibitors of ER stress-induced XBP1 activation II. Structure elucidation

Kawamura T., Tashiro E., Shindo K., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  61 ( 5 ) 312 - 317 2008.05

Research paper (scientific journal), Accepted,  ISSN  00218820

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Four novel triene-ansamycin group compounds, quinotrierixin, demethyltrienomycin A, demethyltrienomycin B and demethyltrienomycinol, were isolated from the culture broth of Streptomyces sp. PAE37 as inhibitors of ER stress-induced XBP1 activation. The structures of quinotrierixin, demethyltrienomycin A, demethyltrienomycin B and demethyltrienomycinol were determined on the basis of their spectroscopical and chemical properties. All of four possessed 21-membered macrocyclic lactams including triene moieties. © Japan Antibiotics Research Association.

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Identification and characterization of mouse type II platelet-derived growth factor receptor α transcript

Minato Y., Nihei Y., Kodama Y., Tashiro E., Kanai M., Imoto M.

Bioscience, Biotechnology and Biochemistry (Bioscience, Biotechnology and Biochemistry)  72 ( 3 ) 759 - 766 2008.04

Research paper (scientific journal), Accepted,  ISSN  09168451

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Recently, we identified a novel human platelet-derived growth factor (PDGF) receptor α mRNA (type II) that differs from the known human PDGF receptor α mRNA (type I) only in its 5′-untranslated region and is regulated by E2F-1. In this study, we determined the nucleotide sequence of the mouse type II PDGF receptor α mRNA and found that the expression of this transcript was regulated by E2F-1 through an E2F-1-responsive sequence located +711/+718 downstream of the transcription start site of the type I transcript. Furthermore, we observed embryo-predominant expression of the type II transcript.

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Discovery of incednine as a potent modulator of the anti-apoptotic function of Bcl-xL from microbial origin

Futamura Y., Sawa R., Umezawa Y., Igarashi M., Nakamura H., Hasegawa K., Yamasaki M., Tashiro E., Takahashi Y., Akamatsu Y., Imoto M.

Journal of the American Chemical Society (Journal of the American Chemical Society)  130 ( 6 ) 1822 - 1823 2008.02

Research paper (scientific journal), Accepted,  ISSN  00027863

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Anti-apoptotic oncoproteins Bcl-2 and Bcl-xL are overexpressed in many cancers and play a crucial role in cancer initiation, progression, and resistance to chemotherapy. Therefore, the discovery of a functional inhibitor for these proteins and improved understanding of the molecular mechanisms of these proteins will be an aid to novel anti-tumor therapies. Here, using chemical-genetic cell-based screening, we have discovered a chemically and biologically unique substance, incednine, as a novel functional modulator of Bcl-2/Bcl-xL from the fermentation broth of Streptomyces sp. ML693-90F3. This compound was isolated as a HCl salt by solvent extraction and using centrifugal liquid-liquid partition chromatography. Its structure was elucidated by spectroscopic analysis, X-ray crystallographic analysis, and computational studies. Incednine has a molecular formula, C42H63N3O8, and consists of a novel skeletal structure, enol-ether amide in a 24-membered macrolactam core, with two aminosugars. Bcl-xL-overexpressing Ms-1 cells displayed resistance to several anti-tumor agents; however, anti-tumor agent-induced cell death was observed only when cells were treated with incednine. Overexpression of Bcl-xL inhibited cell death in Bax-overexpressing HEK293T cells by forming a complex with Bax, whereas Bcl-xL failed to inhibit cell death in the presence of incednine without affecting the heterodimerization of Bcl-xL and Bax. These findings suggest that incednine serves as a potent modulator of the anti-apoptotic Bcl-2/Bcl-xL distinct from the other known Bcl-2 inhibitors and could provide a chemical probe to study the underlying mechanisms of Bcl-2/Bcl-xL. Copyright © 2008 American Chemical Society.

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Synthesis and antitumor activity of combretastatin D-4

Uno K., Tanabe T., Ogamino T., Okada R., Imoto M., Nishiyama S.

Heterocycles (Heterocycles)  75 ( 2 ) 291 - 296 2008.02

Research paper (scientific journal), Accepted,  ISSN  03855414

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Combretastatin D-4 1 was synthesized using electrochemical dimerization of phenols as the key step. Anodic oxidation of bromochloro-phenol 7 provided the corresponding diaryl ether 8. Manipulation of the two hydroxyl groups differenciated, provided the seco-acid, which on Mitsunobu reaction gave the target molecule. Its biological assay showed new potent inhibitory activity against cellular proliferation of human HT-29 colon carcinoma cells. © 2008 The Japan Institute of Heterocyclic Chemistry.

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Transcriptional regulation of a new variant of human platelet-derived growth factor receptor α transcript by E2F-1

Minato Y., Tashiro E., Kanai M., Nihei Y., Kodama Y., Imoto M.

Gene (Gene)  403 ( 1-2 ) 89 - 97 2007.11

Research paper (scientific journal), Accepted,  ISSN  03781119

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Platelet-derived growth factors (PDGFs) and their receptors play an important role in cell proliferation, angiogenesis, and differentiation during normal development, and have also been implicated in tumorigenesis. In this study, we identified a novel variant of human PDGF receptor α mRNA (type II), which contains the same open reading frame as the known PDGF receptor α mRNA (type I) but a different 5′-untranslated region (5′-UTR). The 5′-UTR of the type II transcript was identified as a 363-bp exon located in intron 1 at position + 1210 to + 1572 relative to the transcriptional initiation site of the type I transcript. This type II transcript was expressed in a subset of human cell lines, such as MG-63 and MNNG/HOS cells. Moreover, transcription of the type II, but not the type I, was regulated by E2F-1 through an E2F-1-responsive site located at position + 1086/+ 1093 downstream of the transcriptional initiation site of the type I transcript. Furthermore, epigenetic modulation might be involved in the expression of the type II transcript. Our findings provide new insights into the regulatory mechanism of PDGF receptor α transcription in normal and tumor cells. © 2007 Elsevier B.V. All rights reserved.

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[Development of farnesyltransferase inhibitor for anti-cancer drugs].

Takemoto Y, Imoto M

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 52 ( 13 Suppl ) 1713 - 8 2007.10

Research paper (scientific journal), Accepted,  ISSN  0039-9450

Development of farnesyltransferase inhibitor for anti-cancer drugs

Takemoto Y., Imoto M.

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme (Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme)  52 ( 13 Suppl ) 1713 - 1718 2007.10

Research paper (scientific journal), Accepted,  ISSN  00399450

Trierixin, a novel inhibitor of ER stress-induced XBP1 activation from Streptomyces sp. I. Taxonomy, fermentation, isolation, and biological activities

Tashiro E., Hironiwa N., Kitagawa M., Futamura Y., Suzuki S., Nishio M., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  60 ( 9 ) 547 - 553 2007.09

Research paper (scientific journal), Accepted,  ISSN  00218820

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In the course of screening for an inhibitor of ER stress-induced XBP1 activation, we isolated a new member of the triene-ansamycin group compound, trierixin, from a culture broth of Streptomyces sp. AC 654. Trierixin was purified by column chromatography on silica gel and by HPLC. The molecular formula of trierixin is C37H52N2O8S. Trierixin inhibited thapsigargin-induced XBP1-luciferase activation in HeLa/XBP1-luc cells and endogenous XBP1 splicing in HeLa cells with an IC 50 of 14 ng/ml and 19 ng/ml, respectively. Moreover, in the process of isolating trierixin, we isolated structurally related mycotrienin II and trienomycin A as inhibitors of ER stress-induced XBP1 activation from a culture broth of a trierixin-producing strain. This study provides the first observation that triene-ansamycins have a novel inhibitory effect against XBP1 activation. © Japan Antibiotics Research Association.

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Trierixin, a novel inhibitor of ER stress-induced XBP1 activation from Streptomyces sp. II. Structure elucidation

Futamura Y., Tashiro E., Hironiwa N., Kohno J., Nishio M., Shindo K., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  60 ( 9 ) 582 - 585 2007.09

Research paper (scientific journal), Accepted,  ISSN  00218820

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Trierixin, a new member of the triene-ansamycin group, has been isolated from the fermentation broth of Streptomyces sp. AC654 as an inhibitor of ER stress-induced XBP1 activation. The structure of trierixin was determined on the basis of its spectroscopical and chemical properties. Trierixin possessed a 21-membered macrocyclic lactam, which contains a methylthio-benzenediol structure, and a cyclohexanecarbonylalanine moiety. Trierixin is thus elucidated as 21-thiomethylmycotrienin II. © Japan Antibiotics Research Association.

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Functions of cyclin D1 as an oncogene and regulation of cyclin D1 expression

Tashiro E., Tsuchiya A., Imoto M.

Cancer Science (Cancer Science)  98 ( 5 ) 629 - 635 2007.05

Research paper (scientific journal), Accepted,  ISSN  13479032

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Cyclin D1 binds to the Cdk4 and Cdk6 to form a pRB kinase. Upon phosphorylation, pRB loses its repressive activity for the E2F transcription factor, which then activates transcription of several genes required for the transition from the G1- to S-phase and for DNA replication. The cyclin D1 gene is rearranged and overexpressed in centrocytic lymphomas and parathyroid tumors and it is amplified and/or overexpressed in a major fraction of human tumors of various types of cancer. Ectopic overexpression of cyclin D1 in fibroblast cultures shortens the G1 phase of the cell cycle. Furthermore, it has been demonstrated that introduction of an antisense cyclin D1 into a human carcinoma cell line, in which the cyclin D1 gene is amplified and overexpressed, causes reversion of the malignant phenotype. Thus, increased expression of cyclin D1 can play a critical role in tumor development and in maintenance of the malignant phenotype. However, it is insufficient to confer transformed properties on primary or established fibroblasts. In this review, we summarize the role of cyclin D1 on tumor development and malignant transformation. In addition, our chemical biology study to understand the regulatory mechanism of cyclin D1 transcription is also reviewed. © 2007 Japanese Cancer Association.

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Involvement of protein phosphatase 2A nuclear accumulation and subsequent inactivation of activator protein-1 in leptomycin B-inhibited cyclin D1 expression

Tsuchiya A., Tashiro E., Yoshida M., Imoto M.

Oncogene (Oncogene)  26 ( 11 ) 1522 - 1532 2007.03

Research paper (scientific journal), Accepted,  ISSN  09509232

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Leptomycin B (LMB) is a Streptomyces metabolite that causes the specific inhibition of the nuclear export of proteins containing a nuclear export signal (NES). LMB was reported to inhibit cell cycle progression in fission yeast and mammalian cells, however, the mechanism underlying LMB-induced cell cycle arrest is still obscure. In this study, we found that in serum-starved NIH3T3 cells, LMB inhibited serum-induced cyclin D1 expression at the level of transcription. However, this inhibition was reversed by inhibitors of protein phosphatase 2A (PP2A). Furthermore, we found that PP2A accumulated in the nucleus upon treatment with LMB. The finding prompted us to identify the functional NES in PP2A catalytic subunit α. These results indicated that LMB inhibited the chromosomal region maintenance 1 (CRM1)-dependent nuclear export of PP2A, resulting in sustained dephosphorylation in the nucleus. Although phosphorylation of c-Jun at Ser-63 is required for activator protein 1 (AP-1)-dependent expression of cyclin D1, it decreased in LMB-treated cells compared to untreated cells. Moreover, the inhibitors of PP2A restored the levels of c-Jun phosphorylated at Ser-63. We propose that inhibition of cyclin D1 expression by LMB is mediated by the LMB-induced nuclear accumulation of PP2A, leading to sustained dephosphorylation of c-Jun at Ser-63, which leads to inactivation of the transcription of the AP-1-responsive cyclin D1 gene. © 2007 Nature Publishing Group All rights reserved.

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Synthesis of gerfelin and related analogous compounds

Islam M., Kitagawa M., Imoto M., Kitahara T., Watanabe H.

Bioscience, Biotechnology and Biochemistry (Bioscience, Biotechnology and Biochemistry)  70 ( 10 ) 2523 - 2528 2006.11

Research paper (scientific journal), Accepted,  ISSN  09168451

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Gerfelin, an inhibitor of human geranylgeranyl diphosphate (GGPP) synthase that has been isolated from a culture broth of Beauveria felina QN22047, was synthesized in 4 and 3 steps starting from 2,4-dihydroxy-6-methylbenzoic acid and 3,4,5-trihydroxytoluene, respectively. An effective ligand, 2-(di-tert-butyl-phosphino) biphenyl, was used in the palladium-catalyzed diaryl ether-forming reaction. Five analogous compounds of gerfelin were also synthesized for a study of the structure-activity relationship.

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The education system for chemical biology in Japanese universities.

Osada H., Hagiwara M., Toshima K., Imoto M.

ACS chemical biology (ACS chemical biology)  1 ( 8 ) 492 - 494 2006.09

Research paper (scientific journal), Accepted,  ISSN  1554-8929

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Suppression of multidrug resistance by migrastatin

Takemoto Y., Tashiro E., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  59 ( 7 ) 435 - 438 2006.07

Research paper (scientific journal), Accepted,  ISSN  00218820

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Migrastatin (MGS) is a Streptomyces metabolite that inhibits cancer cell migration. In this study, we found that MGS also enhanced the cytotoxicity of vinblastine, vincristine, and taxol in P-glycoprotein-overexpressing VJ-300 cells and P388/VCR cells. Furthermore, MGS increased the intracellular concentration of labeled vinblastine, vincristine, and taxol in both VJ-300 cells and P388/VCR cells. P-glycoprotein was photolabeled with [ 3H]azidopine, but this photolabeling was significantly inhibited in the presence of MGS. These results indicated that MGS directly interacts with and inhibits P-glycoprotein, thereby sensitizing drug-resistant cells to anticancer drugs. © Japan Antibiotics Research Association.

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Synthesis of verbenachalcone congeners and their biological assessment against activation of the NGF-mediated neurite outgrowth of PC12D cells' activity

Tanabe T., Ogamino T., Shimizu Y., Imoto M., Nishiyama S.

Bioorganic and Medicinal Chemistry (Bioorganic and Medicinal Chemistry)  14 ( 8 ) 2753 - 2762 2006.04

Research paper (scientific journal), Accepted,  ISSN  09680896

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Synthesis of the verbenachalcone derivatives 3-5 involving littorachalcone 2 from diaryl ether 7 enabled an SAR study of enhancement activity against the NGF-mediated neurite outgrowth from PC12D cells. Littorachalcone 2 and o-deoxyverbenachalcone 5 showed similar activity to that of verbenachalcone 1. © 2005 Elsevier Ltd. All rights reserved.

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GSK-3β directly phosphorylates and activates MARK2/PAR-1

Kosuga S., Tashiro E., Kajioka T., Ueki M., Shimizu Y., Imoto M.

Journal of Biological Chemistry (Journal of Biological Chemistry)  280 ( 52 ) 42715 - 42722 2005.12

Research paper (scientific journal), Accepted,  ISSN  00219258

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In Alzheimer disease (AD), the microtubule-associated protein tau is found hyperphosphorylated in paired helical filaments. Among many phosphorylated sites in tau, Ser-262 is the major site for abnormal phosphorylation of tau in AD brain. The kinase known to phosphorylate this particular site is MARK2, whose activation mechanism is yet to be studied. Our first finding that treatment of cells with LiCl, a selective inhibitor of another major tau kinase, glycogen synthase kinase-3β (GSK-3β), inhibits phosphorylation of Ser-262 of tau led us to investigate the possible involvement of GSK-3β in MARK2 activation. In vitro kinase reaction revealed that recombinant GSK-3β indeed phosphorylates MARK2, whereas it failed to phosphorylate Ser-262 of tau. Our further findings led us to conclude that GSK-3β phosphorylates MARK2 on Ser-212, one of the two reported phosphorylation sites (Thr-208 and Ser-212) found in the activation loop of MARK2. Down-regulation of either GSK-3β or MARK2 by small interfering RNAs suppressed the level of phosphorylation on Ser-262. These results, respectively, indicated that GSK-3β is responsible for phosphorylating Ser-262 of tau through phosphorylation and activation of MARK2 and that the phosphorylation of tau at this particular site is predominantly mediated by a GSK-3β-MARK2 pathway. These findings are of interest in the context of the pathogenesis of AD. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Chemistry and biology of moverastins, inhibitors of cancer cell migration, produced by Aspergillus

Takemoto Y., Watanabe H., Uchida K., Matsumura K., Nakae K., Tashiro E., Shindo K., Kitahara T., Imoto M.

Chemistry and Biology (Chemistry and Biology)  12 ( 12 ) 1337 - 1347 2005.12

Research paper (scientific journal), Accepted,  ISSN  10745521

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Cancer cell migration is a required step in cancer metastasis. We screened for inhibitors of cancer cell migration of microbial origin, and obtained moverastin, a member of the cylindrol family, from Aspergillus sp. F7720. However, the results of an NMR spectroscopic analysis raised the possibility that moverastin is a mixture of two diastereomers. Separation of the C-10 epimers of synthetic moverastin and a bioassay revealed that both diastereomers (moverastins A and B) had inhibitory effects on cell migration. Furthermore, we demonstrated that moverastins A and B inhibited FTase in vitro, and they also inhibited both the membrane localization of H-Ras and the activation of the PI3K/Akt pathway in EC17 cells. Thus, moverastins inhibited the migration of tumor cells by inhibiting the farnesylation of H-Ras, and subsequent H-Ras-dependent activation of the PI3K/Akt pathway. ©2005 Elsevier Ltd All rights reserved.

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Involvement of nuclear accumulation of heat shock protein 27 in leptomycin B-induced apoptosis in HeLa cells

Tsuchiya A., Tashiro E., Yoshida M., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  58 ( 12 ) 810 - 816 2005.12

Research paper (scientific journal), Accepted,  ISSN  00218820

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Leptomycin B (LMB) is a Streptomyces metabolite that inhibits the chromosomal region maintenance (CRM)1-dependent nuclear export of proteins. It also induces apoptosis in several types of cancer cells, by a yet undefined mechanism. We used a functional proteomics approach to delineate the pathways and mediators involved in LMB-induced apoptosis in HeLa cells. Using two-dimensional gel electrophoresis, we searched for proteins accumulated in the nuclei of HeLa cells upon LMB treatment. Among such proteins we found prohibitin and Heat shock protein (Hsp)27, identified by peptide mass fingerprinting and mass spectrometry. Immunocytochemistry was carried out to confirm the LMB-induced nuclear accumulation of these two proteins in HeLa cells. Furthermore, we found that the cytoplasmic expression of Hsp27, but not prohibitin, partially inhibited LMB-induced apoptosis, indicating that nuclear sequestration of Hsp27 was, at least in part, involved in the apoptosis. © Japan Antibiotics Research Association.

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Mutational analysis of growth arrest and cellular localization of human immunodeficiency virus type 1 Vpr in the budding yeast, Saccharomyces cerevisiae

Nakazawa J., Watanabe N., Imoto M., Osada H.

Journal of General and Applied Microbiology (Journal of General and Applied Microbiology)  51 ( 4 ) 245 - 256 2005.10

Research paper (scientific journal), Accepted,  ISSN  00221260

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Viral protein R (Vpr), one of the accessory gene products of human immunodeficiency virus type 1 (HIV-1), is responsible for the incorporation of a viral genome into the nucleus upon infection. Vpr also arrests the cell cycle and induces apoptosis in infected cells. Similarly, in yeast, Vpr localizes in the nucleus and shows growth inhibitory activity; however, the molecular mechanism of growth inhibition remains unknown. To elucidate this mechanism, several point mutations of Vpr, which are known to perturb several phenotypes of Vpr in mammalian cells, were introduced in the budding yeast, Saccharomyces cerevisiae. For the first time, we found that growth inhibition by Vpr occurred independently of intracellular localization in yeast, as has previously been reported in mammals. We also indentified several amino acid residues, the mutation of which cancels growth inhibitory activity, and/or alters localization, both in yeast and mammalian cells, suggesting the importance of these residues for the phenotypes. Copyright © 2005 by The Microbiology Research Foundation.

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[Regulation of tumor malignancy by inhibitors of posttranslational modification of Ras and Rho family proteins].

Imoto M

Seikagaku. The Journal of Japanese Biochemical Society 77 ( 6 ) 511 - 8 2005.06

Research paper (scientific journal), Accepted,  ISSN  0037-1017

Regulation of tumor malignancy by inhibitors of posttranslational modification of Ras and Rho family proteins

Imoto M.

Seikagaku. The Journal of Japanese Biochemical Society (Seikagaku. The Journal of Japanese Biochemical Society)  77 ( 6 ) 511 - 518 2005.06

Research paper (scientific journal), Accepted,  ISSN  00371017

Synthesis of the naphthalene-derived inhibitors against Cdc25A dual-specificity protein phosphatase and their biological activity

Shimbashi A., Tsuchiya A., Imoto M., Nishiyama S.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  15 ( 1 ) 61 - 65 2005.01

Research paper (scientific journal), Accepted,  ISSN  0960894X

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The synthesis naphthoquinones exhibited stronger activity than the pyranonaphthoquinone-type compound having moderate inhibitory activity against Cdc25A protein phosphatase. The novel naphthalene-type analogues 14 and 18 and the naphthoquinone-type analogues, 8, 9, 15, 16, 19, 21, 22, and 23-28 have been synthesized, and their in vitro Cdc25A phosphatase-inhibitory activity was examined. In assessment of the inhibitory activity, it was revealed that the naphthoquinone core is contributed to the activity, rather than the alkyl side chain. © 2004 Elsevier Ltd. All rights reserved.

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The synthesis and biological activity of pyranonaphthoquione derivatives from Streptomyces sp. and their related substances

Shimbashi A., Tsuchiya A., Imoto M., Nishiyama S.

Bulletin of the Chemical Society of Japan (Bulletin of the Chemical Society of Japan)  77 ( 10 ) 1925 - 1930 2004.10

Research paper (scientific journal), Accepted,  ISSN  00092673

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The total synthesis of a novel pyranonaphthoquinone (2) having Cdc25A phosphatase inhibitory activity is achieved. The key step in this synthetic pathway is the intramolecular Michael addition for construction of the pyran ring system. The inhibitory activity of several derivatives, readily obtained from synthetic intermediates of 2, is assessed. The resulting data indicates that the pyran ring moiety is not a crucial factor for the activity.

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Gerfelin, a novel inhibitor of geranylgeranyl diphosphate synthase from Beauveria felina QN22047. I. Taxonomy, fermentation, isolation, and biological activities

Zenitani S., Tashiro S., Shindo K., Nagai K., Suzuki K., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  56 ( 7 ) 617 - 621 2003.08

Research paper (scientific journal), Accepted,  ISSN  00218820

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A new compound, gerfelin, was isolated from a culture broth of Beauveria felina QN22047. It was purified by column chromatography on silica gel and by HPLC. Gerfelin has the molecular formula C15H14O6. It inhibited synthesis of geranylgeranyl diphosphate, which was mediated by recombinant human geranylgeranyl diphosphate synthase (hGGPP synthase) in vitro. The inhibitory pattern of gerfelin was noncompetitive against isopentenyl diphosphate, and uncompetitive against farnesyl diphosphate.

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Deletion of the BH1 domain of Bcl-2 accelerates apoptosis by acting in a dominant negative fashion

Kawatani M., Imoto M.

Journal of Biological Chemistry (Journal of Biological Chemistry)  278 ( 22 ) 19732 - 19742 2003.05

Research paper (scientific journal), Accepted,  ISSN  00219258

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To investigate the exact biochemical functions by which Bcl-2 regulates apoptosis, we established a stable human small cell lung carcinoma cell line, Ms-1, overexpressing wild-type human Bcl-2 or various deletion and point mutants thereof, and examined the effect of these Bcl-2 mutants on apoptosis induced by antitumor drugs such as camptothecin. Cytochrome c release, caspase-3(-like) protease activation, and apoptosis induced by antitumor drugs were accelerated by overexpression of Bcl-2 lacking a Bcl-2 homology (BH) 1 domain (Bcl-2/ΔBH1), but not by that of BH2, BH3, or BH4 domain-deleted Bcl-2. A similar result was obtained upon the substitution of glycine 145 with alanine in the BH1 domain (Bcl-2/G145A), which failed to interact with either Bax or Bak. Pro-apoptotic Bax and Bak have been known to be activated in response to antitumor drugs, and Bcl-2/G145A as well as Bcl-2/ΔBH1 also accelerated Bax- or Bak-induced apoptosis in HEK293T cells. These two mutants still retained the ability to interact with wild-type Bcl-2 and Bcl-xL, and abrogated the inhibitory effect of wild-type Bcl-2 or Bcl-xL on Bax- or Bak-induced apoptosis. In addition, immunoprecipitation studies revealed that Bcl-2/ΔBH1 and Bcl-2/G145A interrupted the association between wild-type Bcl-2 and Bax/Bak. Taken together, our results demonstrate that Bcl-2/ΔBH1 or Bcl-2/G145A acts as a dominant negative of endogenous anti-apoptotic proteins such as Bcl-2 and Bcl-xL, thereby enhancing antitumor drug-induced apoptosis, and that this dominant negative activity requires both a failure of interaction with Bax and Bak through the BH1 domain of Bcl-2 and retention of the ability to interact with Bcl-2 and Bcl-xL.

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Transmembrane domain of Bcl-2 is required for inhibition of ceramide synthesis, but not cytochrome c release in the pathway of inostamycin-induced apoptosis

Kawatani M., Uchi M., Simizu S., Osada H., Imoto M.

Experimental Cell Research (Experimental Cell Research)  286 ( 1 ) 57 - 66 2003.05

Research paper (scientific journal), Accepted,  ISSN  00144827

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Bcl-2 protein plays important roles in the regulation of apoptosis. However, the exact mechanism by which Bcl-2 blocks apoptosis is still unclear. In the present study, we found that overexpression of Bcl-2 in human small cell lung carcinoma Ms-1 cells inhibited not only the release of cytochrome c from mitochondria into cytosol but also de novo ceramide synthesis induced by inostamycin, a phosphatidylinositol turnover inhibitor. To investigate the correlation between the structure of Bcl-2 and its inhibitory function in inostamycin-induced apoptosis, Ms-1 cells that stably overexpress domain-deletional mutants of Bcl-2 were established. Transmembrane domain-deleted Bcl-2 failed to inhibit inostamycin-induced de novo ceramide synthesis, whereas it inhibited inostamycin-induced cytochrome c release, indicating that anchoring of Bcl-2 to membrane was a requirement for its inhibitory effect on inostamycin-induced ceramide synthesis, but not cytochrome c release. Thus, the deletion mutant of tarnsmembrane domain of Bcl-2 can suppress inostamycin-induced apoptosis by inhibiting cytochrome c release, a downstream event of ceramide synthesis in the pathway of inostamycin-induced apoptosis. We also found that the BH3 and BH4 domains of Bcl-2 were necessary for inhibition of inostamycin-induced apoptosis, and deletion of BH1 or BH2 did not affect the inhibitory effect of Bcl-2 to inostamycin-induced apoptotic events. © 2003 Elsevier Science (USA). All rights reserved.

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A novel action of terpendole E on the motor activity of mitotic kinesin Eg5

Nakazawa J., Yajima J., Usui T., Ueki M., Takatsuki A., Imoto M., Toyoshima Y., Osada H.

Chemistry and Biology (Chemistry and Biology)  10 ( 2 ) 131 - 137 2003.02

Research paper (scientific journal), Accepted,  ISSN  10745521

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To reveal the mechanism of mitosis, the development of M phase-specific inhibitors is an important strategy. We have been screening microbial products to find specific M phase inhibitors that do not directly target tubulins, and rediscovered terpendole E (TerE) as a novel Eg5 inhibitor. TerE did not affect microtubule integrity in interphase, but induced formation of a monoastral spindle in M phase. TerE inhibited both motor and microtubule-stimulated ATPase activities of human Eg5, but did not affect conventional kinesin from either Drosophila or bovine brain. Although terpendoles have been reported as inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT), the Eg5 inhibitory activity of TerE was independent of ACAT inhibition. Taken together, we demonstrate that TerE is a novel Eg5 inhibitor isolated from a fungal strain.

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Overexpression of cyclin D1 contributes to malignancy by up-regulation of fibroblast growth factor receptor 1 via the pRB/E2F pathway

Tashiro E., Maruki H., Minato Y., Doki Y., Weinstein I., Imoto M.

Cancer Research (Cancer Research)  63 ( 2 ) 424 - 431 2003.01

Research paper (scientific journal), Accepted,  ISSN  00085472

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Overexpression of cyclin D1 due to gene rearrangement, gene amplification, or simply increased transcription occurs frequently in several types of human cancers. However, overexpression of cyclin D1 in cell culture system is insufficient, by itself, to cause malignant transformation. In the present study, we found that when rodent fibroblasts that overexpress cyclin D1, but not normal fibroblasts, were treated with basic fibroblast growth factor (bFGF), there was enhanced cell cycle progression, extracellular signal-regulated kinase 2 activation, induction of anchorage-independent growth, and enhanced invasion of a Matrigel barrier. These enhanced responses to bFGF appear to be due to increased expression of fibroblast growth factor receptor 1, at both the mRNA and protein levels, in the cyclin D1-overexpressing cells. We obtained evidence that this increase in fibroblast growth factor receptor 1 expression is mediated through cyclin D1 activation of the pRB/E2F pathway. Taken together, these results suggest that in vivo cyclin D1 overexpression can enhance tumor progression, at least in part, by potentiating the stimulatory efforts of bFGF, which is often produced by stromal cells, and the growth of adjacent tumor cells.

Regulation of FGF receptor-2 expression by transcription factor E2F-1

Tashiro E., Minato Y., Maruki H., Asagiri M., Imoto M.

Oncogene (Oncogene)  22 ( 36 ) 5630 - 5635 2003

Research paper (scientific journal), Accepted,  ISSN  09509232

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Fibroblast growth factors (FGF) and their receptors play an important role in cell proliferation, angiogenesis and embryonal development. In this study, we show that expression of the FGF receptor-2 (FGFR-2) protein is induced in the mid-to-late G1 phase of the cell cycle in serum-starved mouse NIH3T3 cells released from starvation. Transcription of mouse FGFR-2 was activated by E2F-1. Analysis of various mouse FGFR-2 promoter mutant constructs showed that a sequence located +57/+64 downstream of the transcriptional initiation site, related to the consensus E2F-responsive sequence, is necessary for the activation. The promoter activity of the mouse FGFR-2 gene is also positively regulated by E2F-2 and E2F-3, but not by E2F-4 and E2F-5. Moreover, the E2F-1-induced activation of mouse FGFR-2 gene transcription is suppressed by pRB. Taken together, the results demonstrate that FGFR-2 is a new class of targets for E2F, and expression of mouse FGFR-2 in mid-to-late G1 phase would be mediated, at least in part, by the activation of a pRB/E2F pathway. © 2003 Nature Publishing Group.

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Vacuolar-type H⁺-ATPase inhibitory activity of synthetic analogues of the concanamycins: Is the hydrogen bond network involving the lactone carbonyl, the hemiacetal hydroxy group, and the C-19 hydroxy group essential for the biological activity of the concanamycins?

Yoshimoto Y., Jyojima T., Arita T., Ueda M., Imoto M., Matsumura S., Toshima K.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  12 ( 24 ) 3525 - 3528 2002.12

Research paper (scientific journal), Accepted,  ISSN  0960894X

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Synthetic analogue of the concanamycins, which lacks the hydrogen bond network existing in the concanamycin structure, retains vacuolar-type H+-ATPase (V-ATPase) inhibitory activity and induces apoptosis to cancer cells that overexpressing epidermal growth factor receptors (EGFR). © 2002 Elsevier Science Ltd. All rights reserved.

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Overexpression of cyclin DI contributes to malignant properties of esophageal tumor cells by increasing VEGF production and decreasing Fas expression

Shintani M., Okazaki A., Masuda T., Kawada M., Ishizuka M., Doki Y., Weinstein I., Imoto M.

Anticancer Research (Anticancer Research)  22 ( 2 A ) 639 - 647 2002.05

Research paper (scientific journal), Accepted,  ISSN  02507005

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Cyclin D1 is frequently overexpressed in human esophageal cancer. We examined the possible role of cyclin D1 overexpression on specific malignant properties of tumor cells using a series of eight human esophageal cancer cell lines that express different levels of cyclin D1. We did not find a simple correlation between levels of cyclin D1 expression and anchorage-independent growth, production of angiogenic factors, or tumorigenicity in nude mice, suggesting that other factors can influence these parameters. We did, however, obtain evidence that tumorigenicity appeared to require both the capacity for anchorage-independent growth and the production of angiogenic factors. To better assess the specific role of cyclin D1, we stably expressed an antisense cyclin D1 cDNA construct in the tumorigenic cell line TTn. This significantly decreased anchorage-independent growth and VEGF production and led to a loss of tumorigenicity in nude mice. Furthermore, these cells diplayed a marked increase in sensitivity to antitumor agents and to Fas antibody-induced apoptosis. Taken together, these findings suggest that the overexpression of cyclin D1 can confer esophageal cancer cells with enhanced malignancy through increases in anchorage-independent growth and VEGF production, and down-regulation of Fas expression, thus suggesting novel functions of the cyclin D1 protein in tumor progression.

Absolute configuration of migrastatin, a novel 14-membered ring macrolide.

Nakamura H, Takahashi Y, Naganawa H, Nakae K, Imoto M, Shiro M, Matsumura K, Watanabe H, Kitahara T

The Journal of antibiotics 55 ( 4 ) 442 - 4 2002.04

Research paper (scientific journal), Accepted,  ISSN  0021-8820

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Induction of EGF-dependent apoptosis by vacuolar-type H⁺-ATPase inhibitors in A431 cells overexpressing the EGF receptor

Yoshimoto Y., Imoto M.

Experimental Cell Research (Experimental Cell Research)  279 ( 1 ) 118 - 127 2002

Research paper (scientific journal), Accepted,  ISSN  00144827

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The stimulation of human tumor cells overexpressing epidermal growth factor receptor (EGFR) with EGF enhances tumor development and malignancy. Therefore, compounds that modulate the EGF-mediated signal inducing apoptosis in EGFR-overexpressing cells would represent a new class of antitumor drug and might be useful in the treatment of a subset of human tumors. In the course of screening for compounds that induce apoptosis in EGFR-overexpressing human epidermal carcinoma A431 cells from secondary metabolites of microorganisms, we found that vacuolar-type H+-ATPase (V-ATPase) inhibitors, such as concanamycin B and destruxin E, induced apoptosis only when the cells were stimulated with EGF. The EGF-dependent apoptosis by V-ATPase inhibitors was not observed in other types of human tumor cells which do not overexpress EGFR. The apoptosis in A431 cells was inhibited by anti-FasL antibody which neutralized the cytotoxic effect of FasL, indicating that the Fas/FasL system was involved. The expression of cell surface FasL was upregulated by stimulation with EGF and increased further by V-ATPase inhibitors. Moreover, EGF inhibited cytotoxic Fas antibody-induced apoptosis, whereas V-ATPase inhibitors disrupted the protective effect of EGF on apoptosis in A431 cells. Taken together, these results suggested that V-ATPase inhibitors induced EGF-dependent apoptosis in A431 cells, possibly through both the enhancement of EGF-induced cell surface expression of FasL and the disruption of an EGF-induced survival signal. © 2002 Elsevier Science (USA).

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Study of the novel compounds modulating signal transduction system from microbial origin

Imoto M.

Japanese Journal of Antibiotics (Japanese Journal of Antibiotics)  54 ( 3 ) 117 - 125 2001.05

Research paper (scientific journal), Accepted,  ISSN  03682781

微生物由来細胞内情報伝達系に作用する物質の探索研究.

Jpn. J. Antibiot.  ( 54 ) 117-125 (2001) 2001.03

Single Work

バイオの窓 「大学における物取り研究」

バイオサイエンスとインダストリー  ( 59 ) 43(2000) 2001.03

Single Work

Cytostatic effect of inostamycin, an inhibitor of cytidine 5′-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG): Inositol transferase, on oral squamous cell carcinoma cell lines

Baba Y., Tsukuda M., Mochimatsu I., Furukawa S., Kagata H., Nagashima Y., Koshika S., Imoto M., Kato Y.

Cell Biology International (Cell Biology International)  25 ( 7 ) 613 - 620 2001

Research paper (scientific journal), Accepted,  ISSN  10656995

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Inostamycin, which was recently isolated from Streptomyces sp. MH816-AF15 as an inhibitor of cytidine 5′-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG): inositol transferase, caused a G1-phase accumulation in the cell cycle of small cell lung carcinomas. To investigate whether the cytostatic effect of inostamycin is restricted to lung carcinoma cell lines or applicable to other type of cells, we tested five oral squamous cell carcinoma (SCC) cell lines. Cell growth was suppressed in 62.5-125 ng/ml inostamycin in the culture medium in all oral cancer cell lines tested, with non-viable cells being < 1%, indicating inostamycin is cytostatic on SCC cell lines. Decrease in cyclin D1 mRNA and protein expression due to the inostamycin treatment was accompanied by suppression of phosphorylated retinoblastoma susceptibility gene product (pRB-P) levels. Moreover, flow cytometric analysis showed that inostamycin induced an increase in G1/G0 cells (1.2-3.2 fold) over 24 h. These results suggest that inostamycin is a useful agent for tumour dormant cytostatic therapy for oral SCC. © 2001 Academic Press.

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Inostamycin, an inhibitor of cytidine 5'-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG):inositol transferase, suppresses invasion ability by reducing productions of matrix metalloproteinase-2 and -9 and cell motility in HSC-4 tongue carcinoma cell line.

Y. Baba, M. Tsukuda, I. Mochimatsu, S. Furukawa, H. Kagata, Y. Nagashima, N. Sakai, S. Koshika, M. Imoto, & Y. Kato

Clinical and Experimental Metastasis  ( 18 ) 273-279 (2000) 2000.12

Joint Work

Migrastatin, a novel 14-membered lactone from Streptomyces sp. MK929-43F1.

K. Nakae, Y. Yoshimoto, M. Ueda, T. Sawa, Y. Takahashi, H. Naganawa

J. Antibiot.  ( 53 ) 1228-1230 (2000) 2000.11

Joint Work

Migrastatin, a new inhibitor of tumor cell migration from Streptomyces sp. MK929-43F1.

K. Nakae, Y. Yoshimoto, T. Sawa, Y. Homma, M. Hamada, T. Takeuchi

J. Antibiot.  ( 53 ) 1130-1136 (2000) 2000.11

Joint Work

Bcl-2-independent induction of apoptosis by neuropeptide receptor antagonist in human small cell lung carcinoma cells.

Y. Matsumoto, M. Kawatani, S. Simizu, T. Tanaka, M. Takada

Anticancer Research (Anticancer Research)  20 ( 20 ) 3123-3130 (2000) - 3129 2000.08

Research paper (scientific journal), Joint Work,  ISSN  02507005

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The broad-spectrum antagonist of neuropeptide receptor, [D-Arg1, D-Phe5, D-Trp7,9, Leu17]substance P, induced apoptosis selectively in human small cell lung carcinoma (SCLC) cells, which express gastrin-releasing peptide receptor, but not in other types of tumor cells as well as normal cells. The addition of gastrin-releasing peptide or bombesin and the inhibitor of caspase-3 suppressed [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P-induced apoptosis. Moreover, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P-induced apoptosis was not suppressed by Bcl-2 over-expression. Thus, blockage of gastrin-releasing peptide receptor-mediated signaling may provide a novel therapeutic option in SCLC which has become resistant to conventional chemotherapeutic agents.

Involvement of protein kinase C-regulated ceramidegeneration in inostamycin-induced apoptosis.

M. Kawatani, S. Simizu, H. Osada, M. Takada, N. Arber

Exp. Cell Res. (Experimental Cell Research)  259 ( 259 ) 389-397(2000) - 397 2000.07

Research paper (scientific journal), Joint Work,  ISSN  00144827

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Activation of caspases is commonly involved in the apoptosis induced by various anticancer drugs. However, the upstream events leading to the activation of caspases seem to be specific to each anticancer drug. In the present study, we examined the possible involvement of protein kinase C (PKC) and ceramide generation in caspase-3(-like) protease activation induced by inostamycin, a phosphatidylinositol synthesis inhibitor. Treatment of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, suppressed the release of cytochrome c from mitochondria and the activation of caspase-3(-like) proteases in inostamycin-treated cells, but not in other anticancer drug-treated cells. Inostamycin induced the elevation of intracellular ceramide levels, and fumonisin B1, an inhibitor of ceramide synthase, inhibited inostamycin-induced cytochrome c release, caspase-3(-like) protease activation, and apoptosis. Moreover, TPA also inhibited inostamycin-induced ceramide synthesis. Taken together, our results suggest that inostamycin-induced apoptosis is mediated by PKC-regulated ceramide generation, leading to the activation of a caspase cascade. (C) 2000 Academic Press.

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MK800-62F1, a new inhibitor of apoptotic cell death, from Streptomyces diastatochromogenes MK800-62F1. II. Structure elucidation. J. Antibiot. 53, 575-578 (2000)

Y. Yoshimoto, T. Sawa, H. Naganawa, T.Sugai, T. Takeuchi

J. Antibiot.  ( 53 ) 575-578 (2000) 2000.06

Joint Work

Cloning and charcterization of DIP1, a novel protein that is related to the Id family of proteins.

Y. Yao, Y. Doki, W. Jiang, V. S. Venkatraj, D. Warburton, R. M. Santella, B. Lu, L. Yan, X-H Sun, T. Su, J. Luo, and I. B. Weinstein.

Exp. Cell Res.  ( 257 ) 22-32 (2000) 2000.06

Joint Work

MK800-62F1, a new inhibitor of apoptotic cell death, from Streptomyces diastatochromogenes MK800-62F1. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activity.

Y. Yoshimoto, T. Sawa, N. Kinoshita, Y. Homma, M. Hamada, T. Takeuchi

J. Antibiot. (Journal of Antibiotics)  53 ( 53 ) 569-574 (2000) - 574 2000.06

Research paper (scientific journal), Joint Work,  ISSN  00218820

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A new compound, MK800-62F1, was isolated from a cultured broth of Streptomyces diastatochromogenes MK800-62F1. It inhibited H2O2-induced apoptosis in human small cell lung carcinoma Ms-1 cells as well as in human T-cell leukemia Jurkat cells. In addition, MK800-62F1 also inhibited camptothecin-induced apoptosis in Jurkat cells, which was mediated by intracellular H2O2 generation. MK800-62F1 did not exhibit antioxidative activity in vitro, suggesting that inhibition of apoptosis by MK800-62F1 was not due to the scavenging of H2O2, rather it was due to the modulation of the downstream event of H2O2 generation.

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MK800-62F1, a new inhibitor of apoptotic cell death, from Streptomyces diastatochromogenes MK800-62F1. II. Structure elucidation

Yoshimoto Y., Sawa T., Naganawa H., Sugai T., Takeuchi T., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  53 ( 6 ) 575 - 578 2000.06

Research paper (scientific journal), Accepted,  ISSN  00218820

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A new compound, MK800-62F1, was isolated from a cultured broth of Streptomyces diastatochromogenes MK800-62F1. The structure was determined by NMR analysis and degradation experiments.

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Herbimycin A induces G1 arrest through accumulation of p27(Kip1) in cyclin D1-overexpressing fibroblasts

Endo N., Tashiro E., Umezawa K., Kawada M., Uehara Y., Doki Y., Weinstein I., Imoto M.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications)  267 ( 1 ) 54 - 58 2000.01

Research paper (scientific journal), Accepted,  ISSN  0006291X

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The ansamycin antibiotic herbimycin A is a potent tyrosine kinase inhibitor and reduces the growth rate of various types of mammalian cells. When quiescent Rat6 fibroblast cells were treated with herbimycin A, serum-induced expression of cyclin D1 was inhibited, and this was associated with inhibition of G1 phase progression. However, herbimycin A also inhibited serum-induced G1 progression in derivatives of the Rat6 fibroblast cell line that stably overexpress a human cyclin D1 cDNA (R6ccnD1 4 cells), without affecting the expression levels of G1 cyclins. We found that herbimycin A prevented serum-induced downregulation of the cyclin-dependent kinase inhibitor p27(Kip1), thereby leading to inactivation of the protein kinase activity of CDK2. These results suggest that herbimycin A inhibits a tyrosine kinase(s) that plays a role in degradation of the p27(Kop1) protein. (C) 2000 Academic Press.

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Cloning and characterization of DIP1, a novel protein that is related to the id family of proteins

Yao Y., Doki Y., Jiang W., Imoto M., Venkatraj V., Warburton D., Santella R., Lu B., Yan L., Sun X., Su T., Luo J., Weinstein I.

Experimental Cell Research (Experimental Cell Research)  257 ( 1 ) 22 - 32 2000

Research paper (scientific journal), Accepted,  ISSN  00144827

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Using human cyclin D1 as the 'bait' in a yeast two-hybrid system, together with a HL60 cDNA library, we identified a novel human nuclear protein designated DIP1. This protein is expressed in a variety of cell types, and in fibroblasts its level remains constant throughout the cell cycle. However, the level of this protein increases severalfold during the differentiation of HL60 cells. The DIP1 protein can be phosphorylated in vitro by a cellular kinase and this activity reaches its maximum in extracts obtained from cells in the G1 phase of the cell cycle. DIP1 contains a helix- loop-helix motif but lacks an adjacent basic DNA-binding domain, thus resembling the Id family of proteins. The dip1 gene is located on human chromosome 16pl 1.2-12, a locus that is amplified in several types of human cancer. These results suggest that DIP1 may be involved in the control of gene expression and differentiation, but its precise function remains to be determined. (C) 2000 Academic Press.

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Inostamycin, an inhibitor of cytidine 5′-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG): Inositol transferase, suppresses invasion ability by reducing productions of matrix metalloproteinase-2 and -9 and cell motility in HSC-4 tongue carcinoma cell line

Baba Y., Tsukuda M., Mochimatsu I., Furukawa S., Kagata H., Nagashima Y., Sakai N., Koshika S., Imoto M., Kato Y.

Clinical and Experimental Metastasis (Clinical and Experimental Metastasis)  18 ( 3 ) 273 - 279 2000

Research paper (scientific journal), Accepted,  ISSN  02620898

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Inostamycin is an inhibitor of cytidine 5′-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG): inositol transferase. It significantly reduced epidermal growth factor (EGF)-induced in vitro invasion of the tongue carcinoma cell line, HSC-4, through reconstituted basement membrane Matrigel®. Since phosphatidylinositol (PI) 4,5-biphosphate is important for signal transduction through protein kinase C and actin reorganisation, we further examined the effect of inostamycin on production of two matrix metalloproteinases (MMPs), MMP-2 and -9, and on cell motility. Zymographic analysis showed that inostamycin suppressed pro-MMP-2 and pro-MMP-9 levels at a dose-dependent fashion, while MMP-2 activity was not significantly affected. By reverse transcription-polymerase chain reaction, it was found that inostamycin diminished steady state levels of MMP-2 and -9 but not membrane type 1-MMP mRNA expressions. Inostamycin partially blocked both EGF- and phorbol 12-myristate 13-acetate-stimulated pro-MMP- 9 production. A cytoplasmic calcium chelator (BAPTA-AM) dramatically elevated pro- MMP-9 and slightly elevated pro-MMP-2 secretions. EGF-stimulated motility of HSC-4 cells was suppressed by inostamycin treatment along with reduction of actin cytoskeletal reorganisation, filopodia formation and cdc42 expression. These results suggested that inostamycin would be useful for an anti-invasive agent in tongue cancer.

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Migrastatin, a new inhibitor of tumor cell migration from Streptomyces sp. MK929-43F1 taxonomy, fermentation, isolation and biological activities

Nakae K., Yoshimoto Y., Sawa T., Homma Y., Hamada M., Takeuchi T., Imoto M.

Journal of Antibiotics (Journal of Antibiotics)  53 ( 10 ) 1130 - 1136 2000

Research paper (scientific journal), Accepted,  ISSN  00218820

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A new compound, migrastatin, was isolated from a cultured broth of Streptomyces sp. MK929-43F1, as an inhibitor of tumor cell migration. It was purified by column chromatographies on silica gel and Sephadex LH-20 and HPLC. Migrastatin has the molecular formula of C27H39NO7 consisting of 14-membered macrolide and glutarimide moiety. It inhibited spontaneous migration of human esophageal cancer EC17 cells. Migration inhibitory activity of migrastatin was not dependent on cytotoxicity or inhibition of protein synthesis.

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Requirement of caspase-3 (like) protease-mediated hydrogen peroxide production for apoptosis induced by various anticancer drugs.

清水史郎、高田実、梅沢一夫、井本正哉

J. Biol. Chem. (Journal of Biological Chemistry)  273 ( 273 ) 26900 - 26907 1998.10

Research paper (scientific journal), Joint Work,  ISSN  00219258

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作用機構の異なる種々の制癌剤が共通してカスパーゼ３の活性化、NADPH oxidaseの活性化に伴う過酸化水素の生成という過程をへて、アポトーシスを誘導することを報告した。(8ページ)

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Potentiation of paclitaxel cytotoxicity by inostamycin in human small cell lung carcinoma, Ms-1 cells.

清水史郎、田辺圭子、田代悦、高田実、梅沢一夫、井本正哉

Jpn. J. Cancer Res. (Japanese Journal of Cancer Research)  89 ( 89 ) 970 - 976 1998.09

Research paper (scientific journal), Joint Work,  ISSN  09105050

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イノスタマイシンが小細胞肺癌細胞に対して、タキソールの殺細胞効果を増強することを報告した。(7ページ)

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Caspase-3 activation is not responsible for vinblastine-induced Bcl-2 phosphorylation and G2/M arrest in human small cell lung catcinoma Ms-1 cells.

田代悦、清水史郎、高田実、梅沢一夫、井本正哉

Jpn. J. Cancer Res.  ( 89 )  1998.09

Joint Work

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小細胞肺癌細胞を用いた解析から、ビンブラスチンによるアポトーシスの過程でBcl-2のリン酸化が誘導され、このリン酸化はカスパーゼ3によって阻害されなかった。またビンブラスチンによるG2期停止もカスパーゼ3によって制御されていないことを報告した。(7ページ)

Caspase-3 activation is not responsible for vinblastine-induced Bcl-2 phosphorylation and G2/M arrest in human small cell lung carcinoma Ms-1 cells

Tashiro E., Simizu S., Takada M., Umezawa K., Imoto M.

Japanese Journal of Cancer Research (Japanese Journal of Cancer Research)  89 ( 9 ) 940 - 946 1998.09

Research paper (scientific journal), Accepted,  ISSN  09105050

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Vinblastine arrests cells in the G2/M phase of the cell cycle and subsequently induces cell death by apoptosis. We found that treatment of cells with vinblastine induced phosphorylation of Bcl-2, resulting in the dissociation of Bcl-2 and Bax. Moreover, vinblastine-induced apoptosis was suppressed by an inhibitor of caspase-3, Ac-DEVD-CHO; and a 17-kDa active fragment of caspase-3 was detected following vinblastine treatment, suggesting that caspase-3 is involved in vinblastine-induced apoptosis. However, Ac-DEVD-CHO affected neither vinblastine-induced Bcl-2 phosphorylation nor vinblastine-induced G2/M arrest. Vinblastine caused G2/M arrest prior to apoptosis, whereas vinblastine-induced apoptosis was not dependent on the duration of the G2/M phase. Thus, vinblastine-induced apoptosis might be mediated by the phosphorylation of Bcl-2, resulting in Bcl-2 inactivation, and by subsequent activation of caspase-3.

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Synthesis of fluorescent substrates for protein tyrosine phosphatase assays.

渡辺匠、井本正哉、谷口洋基、木下一彦、梅沢一夫

Bioorg. & Med. Chem. Lett. (Bioorganic and Medicinal Chemistry Letters)  8 ( 8 ) 1301 - 1302 1998.08

Research paper (scientific journal), Joint Work,  ISSN  0960894X

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蛍光標識したチロシンホスファターゼの基質を合成し、簡便なチロシンホスファターゼ測定系を提案した。(2ページ)

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遺伝子の治療への応用　1.癌遺伝子産物(蛋白）を標的とした抗癌剤の開発

井本正哉

臨床消化器内科、  ( 13 )  1998.07

Single Work

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癌遺伝子産物（蛋白）を標的とした抗癌剤について概説し今後の開発における展望を述べた。(7ページ)

Inhibition of cyclin D1 expression and induction of apoptosisby inostamycin in small cell lung carcinoma cells.

井本正哉、田辺圭子、清水史郎、田代悦、高田実、梅沢一夫

Jpn. J. Cancer Res. (Japanese Journal of Cancer Research)  89 ( 89 ) 315 - 322 1998.03

Research paper (scientific journal), Joint Work,  ISSN  09105050

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ホスファチジルイノシトール代謝回転阻害剤イノスタマイシンが、小細胞肺癌細胞に対して、サイクリンD1の発現を抑制しG1期停止を引き起こし、更にアポトーシスを誘導することを報告した。(8ページ)

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最新の癌治療分子標的 a)細胞周期

井本正哉

化学療法の領域、  ( 14 )  1998.03

Single Work

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細胞周期を制御する低分子化合物について概説した。(7ページ)

Induction of hydrogen peroxide production and bax expression by caspase- 3(-like) proteases in tyrosine kinase inhibitor-induced apoptosis in human small cell lung carcinoma cells

Simizu S., Umezawa K., Takada M., Arber N., Imoto M.

Experimental Cell Research (Experimental Cell Research)  238 ( 1 ) 197 - 203 1998.01

Research paper (scientific journal), Accepted,  ISSN  00144827

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In our previous studies (S. Simizu, et al., 1996, Cancer Res. 56, 4978- 4982), we reported that apoptosis of human small cell lung carcinoma (SCLC) cells induced by protein tyrosine kinase inhibitors, such as erbstatin and herbimycin A, was mediated by H2O2 via a newly synthesized protein(s). In the present study, we demonstrated that induction of apoptosis by erbstatin resulted in activation of caspase-3(like) proteases, which are interleukin- 1β-converting enzyme family proteases (caspases) and that inhibition of these protease activities reduced the extent of cell death and H2O2 generation. We also demonstrated that expression of apoptotic protein Bax was induced by erbstatin. Erbstatin-induced Bax expression was inhibited by the inhibitor of caspase-3(-like) proteases. These results indicate that generation of intracellular H2O2 and Bax expression in tyrosine kinase inhibitor-induced apoptosis were modulated by the activation of caspase-3-(- like) proteases in SCLC cells.

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Induction of hydrogen peroxide production and Bax expression by CPP32(-like) proteases in tyrosine kinase inhibitor-induced apoptosis in human small cell lung carcinoma cells.

清水史郎、梅沢一夫、高田実、N. Arber、井本正哉

Exp. Cell Res.  ( 238 )  1998.01

Joint Work

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チロシンキナーゼ阻害剤アーブスタチンによる小細胞肺癌細胞のアポトーシス誘導過程において生成する過酸化水素やBaxの発現はカスパーゼ3によって制御されていることを報告した。(7ページ)

細胞周期を調節する生理活性物質

井本正哉、梅沢一夫

組織培養工学、  ( 23 )  1997.12

Joint Work

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細胞周期を調節する生理活性物質について概説した。(3ページ)

Screening of phosphatidylinositol turnover inhibitors and regulation of cell cycle progression

Umezawa K., Deguchi A., Imoto M.

Japanese Journal of Cancer and Chemotherapy (Japanese Journal of Cancer and Chemotherapy)  24 ( 11 ) 1578 - 1584 1997.11

Research paper (scientific journal), Accepted,  ISSN  03850684

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Phosphatidylinositol (PI) turnover is considered to be involved in the regulation of cell growth. The enzymes for PI turnover include phospholipase C (PLC), PI4-kinase and PI synthase. We have isolated pholipeptin and fluvirucin B2 from microorganisms and akaterpin from a marine sponge as PLC γ inhibitors. We also isolated echiguanines from Streptomyces as P14-kinase inhibitors. Since echiguanines did not inhibit the enzyme in situ, we synthesized their ribosylated derivatives that were effective in cultured cells. We previously isolated inostamycin from Streptomyces as an inhibitor of PI synthase. We found that inostamycin induced G1 block in cycling NRK cells. Inostamycin inhibited the serum-induced S-phase induction in quiescent NRK cells. Inostamycin was found to decrease serum-induced expression of cyclin D and cyclin E, without inhibiting the activation of MAP kinase. It also inhibited serum-induced activation of CDK2 and phosphorylation of pRB. Thus, PI synthesis was suggested to be involved in regulation of serum- induced S-phase induction by modulating G1 cyclin expression.

Effect of cyclin D1 overexpression on G1- progression related events.

井本正哉、土岐裕一郎、W. Jiang, E. K-H. Han,I.B. Weinstein

Exp. Cell Res.  ( 236 )  1997.11

Joint Work

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サイクリンD1を強制発現させたRat6細胞を用いて、サイクリンD1の過剰発現がもたらすG1期進行の異常の機構を解析した。(8ページ)

Effects of cyclin D1 overexpression on G1 progression-related events

Imoto M., Doki Y., Jiang W., Han E., Weinstein I.

Experimental Cell Research (Experimental Cell Research)  236 ( 1 ) 173 - 180 1997.10

Research paper (scientific journal), Accepted,  ISSN  00144827

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In previous studies (W. Jiang, S.M. Kahn, P. Zhou, Y.-J. Zhang, A.M. Cacace, A.S. Infance, Y. Doi, R.M. Santella, and I.B. Weinstein 1993, Oncogene 8, 3447-3457) we reported that stable overexpression of cyclin D1 in R6 rat embryo fibroblasts shortens the G1 phase and impairs growth control. In the present study we examined the effects of cyclin D1 overexpression on other events involved in the G1 to S progression, utilizing the overexpressor cell line R6-ccnD1. We found that when compared to R6 control cells, serum- starved quiescent R6-ccnD1 cells had not only increased levels of the cyclin D1 protein but also increased levels of the cyclin E protein. The latter protein was complexed to phosphorylated cyclin-dependent kinase 2 (CDK2). However, in quiescent serum-starved R6-ccnD1 cells this cyclin E-CKD2 complex lacked in vitro kinase activity due to the presence of a heat-stable inhibitory activity, apparently reflecting the inhibitory effects of the CDK inhibitors (CDK1s) p21(WAF1) and p27(KIP1). Serum stimulation of the quiescent R6-ccnD1 cells was associated with a loss of this inhibitory activity and a decrease in the levels of the latter two proteins, as the cells progressed through the G1 phase. On the other hand, serum stimulation of the control R6 cells was associated with both induction of cyclin E and increased levels of phosphorylated CDK2 proteins and decreased levels of p21(WAF1) and p27(KIP1), as the cells progressed through the G1 phase. Thus, even though over-expression of cyclin D1 can induce the expression of cyclin E and phosphorylated CDK2, premature activation of cyclin E-CDK2 kinase activity in quiescent cells or during progression through G1 appears to be blocked by CDK1s. Nevertheless, the R6ccnD1 cells have a shorter G1 phase than the control cells presumably due to the high levels of both cyclin D1 and cyclin E. Taken together, these results indicate that overexpression of cyclin D, which is frequently seen in human tumors, can have complex effects on the expression of other genes that control cell cycle progression.

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細胞周期と抗がん剤

吉田稔、井本正哉、堀之内末治

BIO Clinica  ( 12 )  1997.10

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抗癌剤の細胞周期に及ぼす影響について概説した。(4ページ)

Enhancement or induction of neurite formation by a protein tyrosine phosphatase inhibitor, 3,4-dephostatin, in growth factor-treated PC12h cells

Fujiwara S., Watanabe T., Nagatsu T., Gohda J., Imoto M., Umezawa K.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications)  238 ( 1 ) 213 - 217 1997.09

Research paper (scientific journal), Accepted,  ISSN  0006291X

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We studied the effect of the 3,4 dihydroxy analogue of dephostatin (3,4-dephostatin), an inhibitor of protein-tyrosine phosphatase (PTPase), on the differentiation of rat pheochromocytoma PC12 cells. 3,4-Dephostatin accelerated NGF-induced neurite formation in PC12h cells, a subline of PC12 cells, whereas the inhibitor alone did not induce neurite formation. It sustained the NGF-induced tyrosine phosphorylation of several proteins, most prominently that of mitogen-activated protein (MAP) kinase. EGF alone did not induce differentiation in PC12h cells, but it induced neurite formation in the presence of 3,4-dephostatin. The inhibitor also prolonged EGF-induced tyrosine phosphorylation and activation of MAP kinase. An inactive analogue of dephostatin, 2'-O-methyl-dephostatin showed no effect on either neurite formation or MAP kinase tyrosine phosphorylation in NGF- or EGF-treated PC12h cells. Thus, we demonstrated that the PTPase inhibitor could enhance growth factor-induced differentiation in PC12 cells possibly by sustaining the MAP kinase activity.

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イノシトール代謝回転阻害剤と細胞周期の制御

梅沢一夫、出口敦子、井本正哉

癌と化学療法、  ( 24 )  1997.09

Joint Work

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イノシトールリン脂質代謝回転阻害剤の細胞周期に及ぼす影響について概説した。(7ページ)

Enhancement or induction of neurite formation by a protein tyrosine phosphatase inhibitor, 3,4- dephostatin, in growthfactor-treated PC12 cells.

藤原聰鯉、渡辺匠、長津利治、合田仁、井本正哉、梅沢一夫

Biochem. Biopys. Res. Commun.,  ( 238 )  1997.09

Joint Work

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チロシンホスファターゼ阻害剤デフォスタチンの誘導体がNGFによるPC12細胞の神経突起伸長を促進したことをを報告した。(5ページ)

Increased expression of the p27KIP1 protein in human esophageal cancer cell lines that over- epress cyclin D1.

土岐裕一郎、井本正哉、E.K-H Han,A. Sgambato, I.B. Weinstein

Carcinigenesis  ( 18 )  1997.06

Joint Work

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ヒト食道癌において、サイクリンD1の過剰発現が観察されるが、サイクリンD1の過剰発現は、同時にp27Kip1の発現も上昇することを報告した。(10ページ)

Increased expression of the p27(KIP1) protein in human esophageal cancer cell lines that over-express cyclin D1

Doki Y., Imoto M., Han E., Sgambato A., Weinstein I.

Carcinogenesis (Carcinogenesis)  18 ( 6 ) 1139 - 1148 1997.06

Research paper (scientific journal), Accepted,  ISSN  01433334

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In the present study we have characterized eight human esophageal squamous carcinoma cell lines for levels of expression of cyclins D1, E, A and B1; CDKs 1, 2 and 4; the CDK inhibitors p16(INK4), p21(WAF1), and p27(KIP1); the retinoblastoma (Rb) protein; and in vitro CDK2- and CDK4-associated kinase activity; and also compared the growth properties of these cell lines. The level of the cyclin D1 protein varied by over 30-fold amongst the eight cell lines. The high level in two cell lines was associated with amplification of this gene, but in three cell lines it was due to post-transcriptional events. Amongst the eight cell lines there was a significant correlation between the levels of cyclin D1, Rb and p27(KIP1) proteins, and CDK4-associated kinase activity. Furthermore, when an exogenous cyclin D1 cDNA was over-expressed in the EC109 cell line by transfection, this led to increased expression of both Rb and p27(KIP1). There was, however, no correlation between the level of cyclin D1 expression and the cell doubling times, duration of the G1 phase, or colony-forming efficiency in agar. Two of the cell lines displayed a high level of the cyclin E protein, low levels of cyclin D1, lacked expression of the Rb protein and expressed high levels of the p16(INK4) protein. One of these cell lines displayed amplification of the latter gene. There was no correlation between the levels of cyclins E or A and in vitro CDK2 kinase activity, but CDK2 kinase activity was inversely correlated with the duration of the G1 phase of the cell cycle. Taken together, these studies indicate marked heterogeneity in the expression of cell cycle-related proteins amongst a series of esophageal carcinoma cell lines. The correlation between the levels of the cyclin D1, Rb and p27(Kip1) proteins suggest the existence of a homeostatic feedback loop between positive and negative acting components of the cell cycle machinery.

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チロシンキナーゼ阻害剤のアポトーシス誘導機構

井本正哉、清水史郎、梅沢一夫

日本農芸化学会誌、  ( 71 )  1997.05

Joint Work

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チロシンキナーゼ阻害剤アーブスタチンのアポトーシス誘導機構について概説した。(4ページ)

Pholipeptin, a novel cyclic lipoundecapeptide from Pseudomonas fluorescens.

宇井英明、三宅俊昭、飯沼寛信、井本正哉、長縄博、服部聖子、浜田雅、竹内富雄、梅沢純夫、梅沢一夫

J. Org. Chem. (Journal of Organic Chemistry)  62 ( 62 ) 103 - 108 1997.01

Research paper (scientific journal), Joint Work,  ISSN  00223263

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新規ホスホリパーゼＣ阻害剤フォリペプチンの構造決定について報告した。(6ページ)

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Induction of apoptotic cell death by tyrosine kinase inhibitor

Imoto M., Simizu S., Umezawa K.

Nippon Nogeikagaku Kaishi (Nippon Nogeikagaku Kaishi)  71 ( 5 ) 523 - 526 1997

Research paper (scientific journal), Accepted,  ISSN  00021407

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Inhibition of G1 cyclin expression in normal rat kidney cells by inostamycin, a phosphatidylinositol synthesis inhibitor.

出口敦子、井本正哉、梅沢一夫

J. Biochem. (Journal of Biochemistry)  120 ( 120 ) 1118 - 1122 1996.12

Research paper (scientific journal), Joint Work,  ISSN  0021924X

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ホスファチジルイノシトール代謝回転阻害剤イノスタマイシンが、G1サイクリンの発現を阻害することによりNRK細胞のG1期進行を阻害することを報告した。(5ページ)

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Synthesis of ribofuranosyl glycosides of echiguanines A and B, inhibitors of phosphatidylinositol 4-kinase

Sanpei K., Saito Y., Imoto M., Umezawa K., Kato K.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  6 ( 21 ) 2487 - 2490 1996.11

Research paper (scientific journal), Accepted,  ISSN  0960894X

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The synthesis of ribofuranosyl glycosides of echiguanines A and B, PI 4-kinase inhibitors, was achieved from 2-amino-4-chloropyrrolo[2,3-d]pyrimidine and 2,3-O-isopropylidene-5-O-(t-butyl) dimethylsilyl-α-D-ribofuranosyl chloride. The ribofuranosyl echiguanine A weakly inhibited PI 4-kinase.

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Involvement of hydrogen peroxide production in erbstatin-induced apoptosis in human small cell lung carcinoma cells

Simizu S., Imoto M., Masuda N., Takada M., Umezawa K.

Cancer Research (Cancer Research)  56 ( 21 ) 4978 - 4982 1996.11

Research paper (scientific journal), Accepted,  ISSN  00085472

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Tyrosine kinase inhibitor, erbstatin, induced morphological apoptosis and DNA fragmentation in human small cell lung carcinoma (SCLC) cells. Erbstatin-induced apoptosis was inhibited by antioxidants, whereas erb- statin-inhibited tyrosine phosphorylation was not affected by them. Erbstatin was shown by means of flow cytometry to induce hydrogen peroxide generation. Furthermore, hydrogen peroxide induced morphological apoptosis and DNA fragmentation in the SCLC cells. We also demonstrated that erbstatin-induced hydrogen peroxide production and DNA fragmentation were partially suppressed by inhibition of protein synthesis. Thus, erbstatin-induced apoptosis would be due to hydrogen peroxide generation via newly synthesized protein.

Involvement of hydrogen peroxide production in erbstatin-induced apoptosis in human small lung carcinoma cells.

清水史郎、井本正哉、益田典幸、高田実、梅沢一夫

Cancer Res.  ( 56 )  1996.11

Joint Work

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チロシンキナーゼ阻害剤アーブスタチンが、小細胞肺癌細胞にアポトーシスを誘導するが、その過程で過酸化水素の生成が重要な役割を果たしていることを報告した。(5ページ)

Synthesis of ribopfuranosyl glycosides of echiguanines A and B, inhibitors of phosphatidy inositol 4-kinase.

三瓶香澄、斉藤義雄、井本正哉、梅沢一夫、加藤国木

Bioorg. & Med. Chem. Lett.  ( 6 )  1996

Joint Work

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ホスファチジルイノシトール4キナーゼ阻害剤エチグアニンAおよびBのグルコシル体を合成し報告した。(4ページ)

細胞情報伝達系に作用する物質の発見と細胞応答の解析

井本正哉

日本農芸化学会誌、  ( 69 )  1995.10

Single Work

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我々の見いだした細胞情報伝達系の阻害剤の単離とそれらを用いた細胞応答の解析の結果を概説した。(7ページ)

Inhibition of phosphatidylinositol-specific phospholipase C activity by fluvirucin B2.

宇井英明、井本正哉、梅沢一夫

J. Antibiot.,  ( 48 )  1995.05

Joint Work

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ホスホリパーゼＣの阻害剤としてフルビルシンB2を放線菌培養液より単離し、それについて報告した。(4ページ)

Induction of erythroid differentiation in leukemic K562 cells by an S-adenosylhomocysteine hydrolase inhibitor, aristeromycin

Mizutani Y., Masuoka S., Imoto M., Kawada M., Umezawa K.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications)  207 ( 1 ) 69 - 74 1995.02

Research paper (scientific journal), Accepted,  ISSN  0006291X

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We have isolated an unusual nucleoside, aristeromycin, from the culture filtrate of Actinomycetes as a compound that induces normal morphology in v-ablts-NIH3T3 cells. Aristeromycin also induced erythroid differentiation in abl-expressing human chronic myelogenous leukaemia K562 cells. It did not affect the amount of Abl or the Abl-associated tyrosine kinase activity in either v-ablts-NIH3T3 or K562 cells. As a potent inhibitor of S-adenosylhomocysteine hydrolase, aristeromycin inhibited methylation of phosphatidylethanolamine to form phosphatidylcholine in K562 cells. Among aristeromycin analogues, the activity to inhibit S-adenosylhomocysteine hydrolase was paralleled with the induction of erythroid differentiation. Thus, aristeromycin inhibits abl functions indirectly, possibly by inhibiting biological methylations. © 1995 Academic Press, Inc.

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Induction of erythroid differentiation in leukaemic K562 cells by an S-adenosylhomocysteine hydrolase inhibitor, aristeromycin.

水谷陽子、舛岡正二郎、井本正哉、川田学、梅沢一夫

Biochem. Biopys. Res. Commun.,  ( 207 )  1995.02

Joint Work

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S-アデノシルホモシステインヒドラーゼ阻害剤アリステロマイシンが、白血病細胞K562を赤芽球様に分化する活性を有することを報告した。(5ページ)

In situ inhibition of protein-tyrosine phosphatase by dephostatin.

井本正哉、出口敦子、早川哲、梅沢一夫

Cell. Pharmacol.  ( 2 )  1995.02

Joint Work

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新規チロシンホスファターゼ阻害剤デフォスタチンを用いて、細胞応答におけるチロシンホスファターゼの関与を報告した。(5ページ)

A novel cyclic lipoundecapeptide, pholipeptin, isolated from Pseudomonas Sp.

宇井英明、三宅俊昭、飯沼寛信、井本正哉、服部聖子、浜田雅、竹内富雄、梅沢純夫、梅沢一夫

tetrohedron Lett. (Tetrahedron Letters)  36 ( 36 ) 7479 - 7480 1995

Research paper (scientific journal), Joint Work,  ISSN  00404039

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ホスホリパーゼＣの阻害剤としてフォリペプチンを単離し、報告した。(2ページ)

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Inhibition of Phosphatidylinositol-specific Phospholipase C Activity by Fluvirucin B<inf>2</inf>

Ui H., Imoto M., Umezawa K.

The Journal of Antibiotics (The Journal of Antibiotics)  48 ( 5 ) 387 - 390 1995

Research paper (scientific journal), Accepted,  ISSN  00218820

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We isolated fluvirucin B2from the culture broth of Streptomyces as an inhibitor of phosphatidylinositol-specific phospholipase C (PI-PLC). It inhibited PI-PLC of A431 cell cytosol with an IC50 of 1.6μg/ml. Fluvirucin B2also inhibited PI-PLC in cultured A431 cells, whereas it did not inhibit phosphatidylinositol synthesis and macromolecular synthesis markedly. It also inhibited epidermal growth factor-induced rapid rounding of A431 cells, in which PI turnover is involved. © 1995, JAPAN ANTIBIOTICS RESEARCH ASSOCIATION. All rights reserved.

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Induction of apoptosis by erbstatin in mouse leukaemia L1210 cells.

清水史郎、井本正哉、梅沢一夫

Biosci. Biotech. Biochem.  ( 58 )  1994.09

Joint Work

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チロシンキナーゼ阻害剤アーブスタチンが、L1210細胞に対し、アポトーシスを誘導することを報告した。(3ページ)

Inhibition of T cell receptor-mediated signal transduction by erbstatin.

井本正哉、中村剛、田中慎一郎、梅沢一夫

Biosci. Biotech. Biochem. (Bioscience, Biotechnology, and Biochemistry)  58 ( 58 ) 1700 - 1701 1994.09

Research paper (scientific journal), Joint Work,  ISSN  09168451

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チロシンキナーゼ阻害剤アーブスタチンが、T細胞の活性化を抑制することを報告した。(2ページ)

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Enhancement of CDP-DG: inositol transferase activity in src- and erbB2-transformed cells.

井本正哉、谷口芳子、藤原宏明、梅沢一夫

Exp. Cell Res., (Experimental Cell Research)  212 ( 212 ) 151 - 154 1994.06

Research paper (scientific journal), Joint Work,  ISSN  00144827

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チロシンキナーゼをコードするerbB2やsrc癌遺伝子で形質転換した細胞では、ホスファチジルイノシトール代謝回転が上昇しており、この活性化にチロシンキナーゼが関与していることを報告した。(4ページ)

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Antitumor effect of piericidin B1 N-oxide.

西岡浩、井本正哉、今岡知美、澤力、竹内富雄、梅沢一夫

J. Antibiot.  ( 47 )  1994.04

Joint Work

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ホスファチジルイノシトール代謝回転阻害剤ピエリシジンB1 N-oxideの制癌活性について報告した。(6ページ)

Involvement of phosphatidylinositol synthesis in the regulation of S-phase induction.

井本正哉、森井隆信、出口敦子、梅沢一夫

Exp. Cell Res. (Experimental Cell Research)  215 ( 215 ) 228 - 233 1994

Research paper (scientific journal), Joint Work,  ISSN  00144827

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正常細胞において、血清で誘導されるS期移行にホスファチジルイノシトール合成が重要な役割を演じている可能性を報告した。(6ページ)

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デフォスタチンの物質特許

 1994

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チロシンホスファターゼ阻害剤デフォスタチンを発見した。

Antitumor effect of piericidin B<inf>1</inf> N-oxide

Nishioka H., Sawa T., Takeuchi T., Imoto M., Imaoka T., Umezawa K.

The Journal of Antibiotics (The Journal of Antibiotics)  47 ( 4 ) 447 - 452 1994

Research paper (scientific journal), Accepted,  ISSN  00218820

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Piericidin B1 N-oxide was isolated from a culture broth of Streptomyces sp. as a novel inhibitor of phosphatidylinositol (PI) turnover. Piericidin B1 N-oxide specifically inhibited orthophosphate labeling of PI induced by epidermal growth factor (EGF) without affecting the formation of phosphatidic acid (PA). Like piericidins A1 and B1, piericidin B1 N-oxide inhibited ATP synthesis in A431 cells; however, the effect of piericidin B1 N-oxide on PI synthesis was stronger than that of piericidins A1 and B1. At the concentration inhibiting PI synthesis, piericidin B1 N-oxide showed no inhibitory effect on DNA, RNA, or protein synthesis. We also demonstrated that piericidin B1 N-oxide reversibly inhibited the growth of A431 cells in situ and suppressed the growth of Ehrlich carcinoma in vivo when administered to mice by intraperitoneal (ip) injection. © 1994, JAPAN ANTIBIOTICS RESEARCH ASSOCIATION. All rights reserved.

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Induction of Apoptosis by Erbstatin in Mouse Leukemia L1210 Cells

Simizu S., Imoto M., Umezawa K.

Bioscience, Biotechnology, and Biochemistry (Bioscience, Biotechnology, and Biochemistry)  58 ( 9 ) 1549 - 1552 1994

Research paper (scientific journal), Accepted,  ISSN  09168451

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Erbstatin, a tyrosine kinase inhibitor, has antitumor activity on L1210 mouse leukemia. We tried to find the mechanism of this cytotoxic effect of erbstatin. Erbstatin increased trypan blue exclusion in cultured LI210 cells. The cytotoxic action of erbstatin was accompanied with shrinking of the cells and internucleosomal DNA fragmentation, characteristics of apoptosis. The DNA fragmentation was induced with other tyrosine kinase inhibitors as well, methyl 2,5-dihydroxycinnamate and herbimycin A. In addition, intracellular tyrosine phosphorylation was inhibited by erbstatin before cell death. Erbstatin-induced DNA fragmentation was prevented by zinc ions, but not by cycloheximide or 12-O-tetradecanoylphorbol 13-acetate (TPA). Thus, erbstatin induced apoptotic cell death in L1210 cells by inhibiting tyrosine kinase. © 1994, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.

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Induction of normal phenotypes in ras-transformed cells by damnacanthal from Morinda citrifolia.

平松朋範、井本正哉、小谷野隆、梅沢一夫

Cancer Lett. (Cancer Letters)  73 ( 73 ) 161 - 166 1993.11

Research paper (scientific journal), Joint Work,  ISSN  03043835

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熱帯植物モリンダシトリフォリアからras癌遺伝子で形質転換した細胞の形態正常化を誘導する物質ダムナカンタールを単離し、報告した。(6ページ)

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Dephostatin, a novel protein tyrosine phosphatase inhibitor produced by Streptomyces. II. Structure determination.

掛谷秀昭、井本正哉、高橋良和、長縄博、竹内富雄、梅沢一夫

J. Antibiot.  ( 46 )  1993.11

Joint Work

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新規チロシンホスファターゼ阻害剤デフォスタチンの構造解析について報告した。(4ページ)

Dephostatin, a novel protein tyrosine phosphatase inhibitor produced by Streptomyces. I.. Taxonomy, Isolation, and Characterization.

井本正哉、掛谷秀昭、澤力、林千草、浜田雅、竹内富雄、梅沢一夫

J. Antibiot. (the journal of antibiotics)  46 ( 46 ) 1342 - 1346 1993.09

Research paper (scientific journal), Joint Work,  ISSN  00218820

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放線菌よりチロシンホスファターゼ阻害剤として新規化合物デフォスタチンを発見し、報告した。(5ページ)

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Dephostatin, a novel protein tyrosine phosphatase inhibitor produced by Streptomyces: Structure determination

Kakeya H., Imoto M., Takahashil Y., Naganawa H., Takeuchi T., Umezawa K.

Journal of Antibiotics (Journal of Antibiotics)  46 ( 11 ) 1716 - 1719 1993.07

Research paper (scientific journal), Accepted,  ISSN  00218820

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Dephostatin, a novel tyrosine phosphatase inhibitor, was isolated from the culture broth of Streptomyces sp. MJ742-NF5. The structure was elucidated to be 2-(N-methyl-N-nitroso)hydroquinone by spectral and chemical analyses of dephostatin and its derivatives. © 1993, JAPAN ANTIBIOTICS RESEARCH ASSOCIATION. All rights reserved.

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Isolation of a novel substrate-competitive tyrosine kinase inhibitor, desmal, from the plant Desmos chinensis.

掛谷秀昭、井本正哉、田端裕二、岩見潤子、松本晴彦、中村一夫、小谷野隆、只野金一、梅沢一夫

FEBS Lett. (FEBS Letters)  320 ( 320 ) 169 - 172 1993.04

Research paper (scientific journal), Joint Work,  ISSN  00145793

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熱帯植物Desmos chinennsisからチロシンキナーゼ阻害剤デスマールを単離し、報告した。(4ページ)

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Involvement of tyrosine kinase in growth factor- inducedphospholipase C activation in NIH3T3 cells.

井本正哉、筋誡郁夫、宇井英明、梅沢一夫

Biochim. Biophys. Acta (Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism)  1166 ( 1166 ) 188 - 192 1993.02

Research paper (scientific journal), Joint Work,  ISSN  00052760

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チロシンキナーゼ阻害剤アーブスタチンが、NIH3T3細胞のPDGFやボンベシンにより誘導されるホスホリパーゼＣの活性化を抑制したことを報告した。(５ページ)

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イノシトールリン脂質代謝回転阻害剤の探索と生物活性

梅沢一夫、井本正哉

実験医学,  ( 11 )  1993

Joint Work

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我々が見いだしたホスファチジルイノシトール代謝回転阻害剤の単離、精製、および生物活性について概説した。(６ページ)

ホスファチジルイノシトール代謝回転阻害剤と細胞周期の制御

井本正哉、梅沢一夫

cell Science  ( 8 )  1992.08

Joint Work

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ホスファチジルイノシトール代謝回転阻害剤であるイノスタマイシンの細胞周期に及ぼす影響について概説した。（7ページ）

Biosynthetic preparation of radioactively labeled erbstatin.

田端裕二、井本正哉、梅沢一夫

J. Antibiot. (The Journal of Antibiotics)  45 ( 45 ) 1382 - 1384 1992.08

Research paper (scientific journal), Joint Work,  ISSN  00218820

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放線菌におけるチロシンキナーゼ阻害剤アーブスタチンの生合成を検討し、その知見を基に放射能ラベル化されたアーブスタチンの合成法を報告した。(3ページ)

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Inhibition of CDP-DG: inositol transferase by inostamycin.

井本正哉、谷口芳子、梅沢一夫

J. Biochem. (Journal of Biochemistry)  112 ( 112 ) 299 - 302 1992.02

Research paper (scientific journal), Joint Work,  ISSN  0021924X

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ホスファチジルイノシトール代謝回転阻害剤であるイノスタマイシンの阻害作用点を解析し、イノスタマイシンがCDP-DG:イノシトールトランスフェラーゼであることを報告した。（4ページ）

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Inhibition of tyrosine kinase and src oncogene functions by stable erbstatin analogues.

堀隆、近藤哲朗、辻友子、井本正哉、梅沢一夫

J. Antibiot. (The Journal of Antibiotics)  45 ( 45 ) 280 - 282 1992.02

Research paper (scientific journal), Joint Work,  ISSN  00218820

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チロシンキナーゼ阻害剤アーブスタチンの安定誘導体がラウス肉腫ウイルスで形質転換した細胞のチロシンキナーゼを阻害しサーク癌遺伝子の機能を阻害することを報告した。(3ページ)

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Inhibition of epidermal growth factor-induced activation of phospholipase C by psi-tectorigenin.

井本正哉、志村直巳、梅沢一夫

J. Antibiot. (Journal of Antibiotics)  44 ( 44 ) 915 - 917 1991.08

Research paper (scientific journal), Joint Work,  ISSN  00218820

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ホスファチジルイノシトール代謝回転阻害剤であるプシテクトリゲニンが、EGFにより誘導されるホスホリパーゼＣの活性化を抑制したことを報告した。(3ページ)

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Induction of morphological change by tyrosine kinase inhibitors in Rous sarcoma virus-transformed rat kidney cells

Umezawa K., Tanaka K., Hori T., Abe S., Sekizawa R., Imoto M.

FEBS Letters (FEBS Letters)  279 ( 1 ) 132 - 136 1991.02

Research paper (scientific journal), Accepted,  ISSN  00145793

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Erbstatin and methyl 2,5-dihydroxycinnamate, related tyrosine kinase inhibitors, induced a morphological change in temperature-sensitive Rous sarcoma virus-transformed rat kidney (RSV15·NRK) that brought the cells close to the morphology of their normal counterpart. Erbstatin did not change the morphology of normal or Kirsten sarcoma virus-transformed rat kidney cells. Erbstatin also inhibited morphological transformation of RSV15·NRK cells induced by a shifting in temperature. Actin stress fibres were observed only in normal cells and not in transformed cells. Erbstatin induced stress fibre organization in transformed cells. Erbstatin and methyl 2.5-dihydroxycinnamate increased fibronectin gene expression in RSV-transformed cells. Thus, tyrosine kinase inhibitors induced normal phenotypes specifically in v-sre-expressing cells. © 1991.

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Suppression of multidrug resistance by inostamycin in cultured human carcinoma KB cells.

川田学、井本正哉、梅沢一夫

J. Cell Pharmacol.  ( 2 )  1991.02

Joint Work

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イノスタマイシンを多剤耐性KB細胞に作用させると、アドリアマイシンによる耐性を克服することを報告した。(5ページ)

Induction of morphological change by tyrosine kinase inhibitors in Rous sarcoma virus-transformed rat kidney celss.

梅沢一夫、田中かろ、堀隆、安部聡子、関沢隆一、井本正哉

FEBS Lett.  ( 278 )  1991.02

Joint Work

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チロシンキナーゼ阻害剤アーブスタチンおよびその誘導体がラウス肉腫ウイルスで形質転換した細胞の形態を正常細胞様に復帰させたことを報告した。(5ページ)

Inhibition by phosphatidylinositol turnover inhibitors of epidermal growth factor-induced rounding of A431 cells.

井本正哉、城頭範子、梅沢一夫

J. Cell Pharmacol.  ( 2 )  1991.01

Joint Work

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A431細胞にEGFを添加するとrapid roundingを引き起こすが、ホスファチジルイノシトール代謝回転阻害剤であるイノスタマイシンやプシテクトリゲニンは、その効果を阻害することを報告した。(5ページ)

Use of Erbstatin as Protein-Tyrosine Kinase Inhibitor

Umezawa K., Imoto M.

Methods in Enzymology (Methods in Enzymology)  201 ( C ) 379 - 385 1991.01

Research paper (scientific journal), Accepted,  ISSN  00766879

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This chapter discusses the use of erbstatin as protein-tyrosine kinase inhibitor. Streptomyces and other microorganisms produce antibiotics, anticancer agents, and enzyme inhibitors as secondary metabolites. Isolation of specific bioactive products in culture broths depends on the assay system. Erbstatin is easily inactivated in calf serum, and more stable analogs of erbstatin have been looked for. Various erbstatin analogs have been synthesized and tested for their inhibitory activity against tyrosine kinase. This analog inhibits tyrosine kinase in vitro, but are not active in vivo, because, being a polar molecule, it would penetrate the cells poorly. © 1991, Elsevier Inc. All rights reserved.

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Use of erbstatin as a protein-tyrosine kinase inhibitor.

梅沢一夫、井本正哉

Methods in enzymology  ( 201 )  1991

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チロシンキナーゼ阻害剤アーブスタチンのスクリーニング、単離、生物活性について概説した。(7ページ)

Inhibition of phosphatidylinositol kinase by toyocamycin.

西岡浩、澤力、浜田雅、志村直巳、井本正哉、梅沢一夫

J. Antibiot. (The Journal of Antibiotics)  43 ( 43 ) 1586 - 1589 1990.12

Research paper (scientific journal), Joint Work,  ISSN  00218820

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ホスファチジルイノシトール　キナーゼの阻害剤をスクリーニングした結果、トヨカマイシンを単離し、更にその間連化合物の阻害活性についても報告した。(4ページ)

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Inhibition of EGF-induced phospholipase C activation in A341 cells by erbstatin, a tyrosine kinase inhibitor

Imoto M., Shimura N., Hideaki U., Umezawa K.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications)  173 ( 1 ) 208 - 211 1990.11

Research paper (scientific journal), Accepted,  ISSN  0006291X

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Erbstatin, a tyrosine kinase inhibitor, inhibited epidermal growth factor (EGF)-induced inositol phosphate production in cultured A431 cells. However, it did not inhibit ATP-induced inositol phosphate production. Cytosolic but not membrane-associated phospholipase C was iactivated by EGF, and erbstatin inhibited enhancement of the phospholipiase C activity in EGF-treated cells. Thus, tyrosine kinase of A431 cells is suggested to be functionally involved in phospholipase C activation. © 1990 Academic Press, Inc.

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Inhibition of EGF-induced phospholipase C activation in A431 cells by erbstatin, a tyrosine kinase inhibitor.

井本正哉、志村直巳、宇井英明、梅沢一夫

Biochem. Biopys. Res. Commun.,  ( 173 )  1990.11

Joint Work

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チロシンキナーゼ阻害剤アーブスタチンが、EGFで誘導されるPLCの活性化を阻害したことより、PLC活性化の過程にチロシンキナーゼが、密接に関与していることを報告した。(3ページ)

癌遺伝子機能発現に作用する微生物二次代謝産物　

梅沢一夫、井本正哉

Cell Science  ( 6 )  1990.09

Joint Work

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我々が見いだした癌遺伝子産物の機能阻害剤について概説した。(8ページ)

Isolation and structure determination of inosta- mycin, a novel inhibitor of phosphatidylinositol turnover.

井本正哉、梅沢一夫、高橋良和、長縄博、飯高洋一、中村光、小泉由美、佐々木由美、浜田雅、澤力、竹内富雄

J. Natl. Prod. (Journal of Natural Products)  53 ( 53 ) 825 - 829 1990.07

Research paper (scientific journal), Joint Work,  ISSN  01633864

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ホスファチジルイノシトール代謝回転阻害物質として、イノスタマイシンを発見し、構造解析について報告した。(5ページ)

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Inhibition of epidermal growth factor-induced DNA synthesis by tyrosine kinase inhibitors.

梅沢一夫、堀隆、田島裕久、井本正哉、一色邦夫、竹内富雄

FEBS Letter (FEBS Letters)  260 ( 260 ) 198 - 200 1990.01

Research paper (scientific journal), Joint Work,  ISSN  00145793

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チロシンキナーゼ阻害剤アーブスタチン誘導体の細胞周期に及ぼす影響を報告した。(3ページ)

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Screening of phosphatidylinositol kinase inhibitors from Streptomyces.

西岡浩、井本正哉、澤力、浜田雅、長縄博、竹内富雄、梅沢一夫

J. Antibiot. (The Journal of Antibiotics)  42 ( 42 ) 823 - 825 1989.05

Research paper (scientific journal), Joint Work,  ISSN  00218820

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ホスファチジルイノシトールキナーゼ阻害物質の探索方法を開発し、報告した。(3ページ)

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Modification of cellular membrane functions by pendolmycin.

梅沢一夫、井本正哉、山下敬、澤力、竹内富雄

J.pn. J. Cancer Res. (Japanese Journal of Cancer Research)  80 ( 80 ) 15 - 18 1989.01

Research paper (scientific journal), Joint Work,  ISSN  09105050

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新規インドールアルカロイド化合物ペンドールマイシンの細胞に対する影響を報告した。(4ページ)

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イノスタマイシンの物質特許

 1989

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イノシトールリン脂質阻害剤イノスタマイシンを発見した。

Isolation of a new indole alkaloid, pendol mycin from Nocardiopsis.

山下敬、井本正哉、一色邦夫、澤力、長縄博、倉沢省伍、Z. Bao-quan、梅沢一夫

J. Natl. Prod.  1988.11

Joint Work

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新規インドールアルカロイド化合物ペンドールマイシンを単離し、構造決定について報告した。(4ページ)

Isolation of a new indole alkaloid, pendolmycin, from nocardiopsis

Yamashita T., Imoto M., Isshiki K., Sawa T., Naganawa H., Kurasawa S., Umezawa K., Zhu B.

Journal of Natural Products (Journal of Natural Products)  51 ( 6 ) 1184 - 1187 1988.11

Research paper (scientific journal), Accepted,  ISSN  01633864

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—A new indole alkaloid, pendolmycin {1}, was isolated from NocardioPsis strain SA1715 as an inhibitor of epidermal-growth-factor-induced phosphatidylinositol turn-over in A431 cells. The structure of pendolmycin is closely related to that of teleocidin B {2} and of lyngbyatoxin A [3]. Pendolmycin may be a biosynthetic intermediate of these alkaloids. © 1988, American Chemical Society. All rights reserved.

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Inhibition of cellular phosphatidylinosito, turnover by psi-tectorigenin

Imoto M., Yamashita T., Sawa T., Kurasawa S., Naganawa H., Takeuchi T., Bao-quan Z., Umezawa K.

FEBS Letters (FEBS Letters)  230 ( 1-2 ) 43 - 46 1988.03

Research paper (scientific journal), Accepted,  ISSN  00145793

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Psi-tectorigenin, an isoflavonoid, was isolated from a culture filtrate of actinomycetes as an inhibitor of epidermal growth factor-induced phosphatidylinositol turnover in cultured A431 cells. It inhibited phosphatidylinositol turnover with an IC50 of about 1 μg/ml; thus, its inhibitory activity was 6-times stronger than that of genistein or orobol. When added to cultured A431 cells psi-tectorigenin inhibited phosphatidylinositol turnover without inhibiting epidermal growth factor receptor tyrosine protein kinase. Thus, psi-tectorigenin is a specific inhibitor of phosphatidylinositol turnover and may be a useful tool for the functional analysis of phosphatidylinositol turnover. © 1988.

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New potent anthracyclines, barminomycins I and II

内田丈士、井本正哉、高橋良和、小田川淳夫、澤力、竜田邦明、長縄博、浜田雅、竹内富雄、梅沢浜夫

J. Antibiot. (The Journal of Antibiotics)  41 ( 41 ) 404 - 408 1988.03

Research paper (scientific journal), Joint Work,  ISSN  00218820

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新規制癌物質バルミノマイシンIとIIを単離し、それらの制癌活性を報告した。(5ページ)

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Inhibition of cellular phosphatidylinositol turnover by psi-tectorigenin.

井本正哉、山下敬、澤力、倉沢省伍、長縄博、竹内富雄、Z. Bao-quan、梅沢一夫

FEBS Letter  ( 230 )  1988.03

Joint Work

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ホスファチジルイノシトール代謝回転阻害物質として、プシテクトリゲニンを単離し、細胞に対する影響について報告した。(4ページ)

In situ inhibition of tyrosine protein kinase by erbstatin.

井本正哉、梅沢一夫、澤力、倉沢省伍、竹内富雄、梅沢浜夫

Biochem. Int. (Biochemistry International)  15 ( 15 ) 989 - 995 1987.11

Research paper (scientific journal), Joint Work,  ISSN  01585231

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チロシンキナーゼ阻害剤アーブスタチンの培養細胞での細胞応答を解析し、チロシンキナーゼの生理的役割を報告した。(7ページ)

Kinetic studies of tyrosine kinase inhibition by erbstatin.

井本正哉、梅沢一夫、一色邦夫、国元節子、澤力、竹内富雄、梅沢浜夫

J. Antibiot. (The Journal of Antibiotics)  40 ( 40 ) 1471 - 1473 1987.10

Research paper (scientific journal), Joint Work,  ISSN  00218820

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チロシンキナーゼ阻害剤アーブスタチンの阻害様式を検討し、報告した。(3ページ)

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Inhibition of tyrosine protein kinase by synthetic erbstatin analogs.

一色邦夫、井本正哉、澤力、梅沢一夫、竹内富雄、梅沢浜夫,土田俊雄、吉岡武男、竜田邦明

J. Antibiot. (The Journal of Antibiotics)  40 ( 40 ) 1209 - 1210 1987.08

Research paper (scientific journal), Joint Work,  ISSN  00218820

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合成されたアーブスタチンの誘導体のチロシンキナーゼ阻害活性と構造活性相関を報告した。(2ページ)

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Effective synthesis of erbstatin and its analogs.

一色邦夫、井本正哉、竹内富雄、梅沢浜夫、土田俊雄、吉岡武男、竜田邦明

J. Antibiot. (Journal of Antibiotics)  40 ( 40 ) 1207 - 1208 1987.08

Research paper (scientific journal), Joint Work,  ISSN  00218820

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チロシンキナーゼ阻害剤アーブスタチンとその誘導体の全合成を報告した。(2ページ)

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Antitumor activity of erbstatin, a tyrosine protein kinase inhibitor.

井本正哉、梅沢一夫、小室圭子、澤力、竹内富雄、梅沢浜夫

Jpn. J. Cancer Res. (Japanese Journal of Cancer Research)  78 ( 78 ) 329 - 332 1987.04

Research paper (scientific journal), Joint Work,  ISSN  09105050

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チロシンキナーゼ阻害剤アーブスタチンの制癌効果を動物実験によって検討し、結果を報告した。(4ページ)

The structure of an epidermal growth factor- receptor kinase inhibitor, erbstatin.

中村光、飯高洋一、井本正哉、一色邦夫、長縄博、竹内富雄、梅沢浜夫

J. Antibiot. (The Journal of Antibiotics)  39 ( 39 ) 314 - 315 1985.02

Research paper (scientific journal), Joint Work,  ISSN  00218820

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新規チロシンキナーゼ阻害剤アーブスタチンを発見し、その構造決定を報告した。(2ページ)

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Studies on a new epidermal growth factor-receptor kinaseinhibitor, erbstatin, produced by MH435-hF3.

梅沢浜夫、井本正哉、澤力、一色邦夫、松田直子、内田丈士、飯沼寛信、浜田雅、竹内富雄

J. Antibiot.  ( 39 )  1985.01

Joint Work

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新規チロシンキナーゼ阻害剤アーブスタチンを発見し、報告した。(4ページ)

新規チロシンキナーゼ阻害剤の物質特許

 1985

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新規チロシンキナーゼ阻害剤アーブスタチンを発見した。

Saquayamycins, new aquayamycin-group antibiotics.

内田丈士、井本正哉、渡辺義範、三浦圭子、土橋淑子、松田直子、澤力、長縄博、浜田雅、竹内富雄、梅沢浜夫

J. Antibiot. (the journal of antibiotics)  38 ( 38 ) 1171 - 1181 1984.09

Research paper (scientific journal), Joint Work,  ISSN  00218820

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新規制癌物質サクワヤマイシンを単離し、それらの制癌活性を報告した。(4ページ)

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New anthracycline antibiotics: Serirubicin and 1-hydroxyserirubicin.

内田丈士、井本正哉、増田徹、 吉本宏、今村寛司、澤力、長縄博、竹内富雄、梅沢浜夫

J. Antibiot. (Journal of Antibiotics)  38 ( 38 ) 795 - 798 1984.06

Research paper (scientific journal), Joint Work,  ISSN  00218820

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新規制癌物質セリルビシンおよび1-ヒドロキシセリルビシンを単離し、それらの制癌活性を報告した。(4ページ)

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New antitumor antibiotics, ditrisarubicins A, B and C.

内田丈士、井本正哉、増田徹、今村寛司、羽鳥洋一、澤力、長縄博、浜田雅、竹内富雄、梅沢浜夫

J. Antibiot.  ( 36 )  1983.08

Joint Work

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新規制癌物質ダイトリサルビシンA,B,Cを単離し、それらの制癌活性を報告した。(4ページ)

Participation and properties of lipoxygenase and formation hydroperoxide lyase in volatile C6-aldehydefrom C18-unsaturated fatty acids in isolated tea chloroplasts.

畑中顯和、梶原忠彦、関谷次郎、井本正哉、井上敏

Plant & Cell Physiol.  ( 23 )  1982.01

Joint Work

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クロロプラストの脂肪酸からC6アルデヒド生成にリポキシゲナーゼとリアーゼが関与していることを報告した。（9ページ）

Participation and properties of lipoxygenase and hydroperoxide lyase in volatile C<inf>6</inf>-aldehyde formation from C<inf>18</inf>-unsaturated. Fatty acids in isolated tea chloroplasts

Hatanaka A., Kajiwara T., Sekiya J., Imoto M., Inouye S.

Plant and Cell Physiology (Plant and Cell Physiology)  23 ( 1 ) 91 - 99 1982.01

Research paper (scientific journal), Accepted,  ISSN  00320781

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Isolated tea chloroplasts utilized linoleic acid, linolenic acid and their 13-hydroperoxides as substrates for volatile C6-aldehyde formation. Optimal pH values for oxygen uptake, hydroperoxide lyase and the overall reaction from C18-fatty acids to C6-aldehydes were 6.3, 7.0 and 6.3, respectively. Methyl linoleate, linoleyl alcohol and γ-linolenic acid were poor substrates for the overall reaction, but linoleic and linolenic acids were good substrates. The 13-hydroperoxides of the above fatty acids and alcohol also showed substrate specificity similar to that of fatty acids. Oxygen uptakes (relative Vmax) with methyl linoleate, linoleyl alcohol, linolenic acid, γ-linolenic acid and arachidonic acid were comparable to or higher than that with linoleic acid. In winter leaves, the activity for C6-aldehyde formation from C18-fatty acids was raduced to almost zero. This was due to the reduction in oxygenation. The findings presented here provide evidence for the involvement of lipoxygenase and hydroperoxide lyase in C6-aldehyde formation in isolated chloroplasts. Copyright © 1982. The Japanese Society of Plant Physiologists.

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Effect of lipolytic acyl hydrolase on the activity for six carbon aldehyde formation in tea chloroplasts.

関谷次郎、梶原忠彦、井本正哉、井上敏、畑中顯和

J. Agric. Food Chem. (Journal of Agricultural and Food Chemistry)  30 ( 30 ) 183 - 185 1982

Research paper (scientific journal), Joint Work,  ISSN  00218561

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クロロプラストをLAHase処理すると脂肪酸の酸化酸化ステップが阻害され、C6アルデヒド生成が抑制されることを報告した。（3ページ）

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