Publications by authors named "Norikazu Nishino"

62 Publications

Improved development of mouse SCNT embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors†.

Biol Reprod 2021 May 12. Epub 2021 May 12.

RIKEN BioResource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074 Japan.

In mammalian cloning by somatic cell nuclear transfer (SCNT), treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit Class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1 to 7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2 to 7.3%). Thus, inhibition of Class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with Class I inhibitors causes a 2-cell developmental block. Therefore, we used Ky-29, with higher selectivity for Class IIa than Class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the 2-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the 1-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.
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http://dx.doi.org/10.1093/biolre/ioab096DOI Listing
May 2021

Transcriptomic analysis of Arabidopsis thaliana plants treated with the Ky-9 and Ky-72 histone deacetylase inhibitors.

Plant Signal Behav 2018 03 22;13(3):e1448333. Epub 2018 Mar 22.

a Plant Genomic Network Research Team , RIKEN Center for Sustainable Resource Science 1-7-22 , Suehiro-cho, Tsurumi-ku, Yokohama , Kanagawa , Japan.

Histone acetylation plays a pivotal role in plant growth and development, and is regulated by the antagonistic relationship between histone acetyltransferase (HAT) and histone deacetylase (HDAC). We previously revealed that some HDAC inhibitors confer high-salinity stress tolerance in plants. In this study, we identified two HDAC inhibitors, namely Ky-9 and Ky-72, which enhanced the high-salinity stress tolerance of Arabidopsis thaliana. Ky-9 and Ky-72 are structurally similar chlamydocin analogs. However, the in vitro inhibitory activity of Ky-9 against mammalian HDAC is greater than that of Ky-72. A western blot indicated that Ky-9 and Ky-72 increased the acetylation levels of histone H3, suggesting they exhibit HDAC inhibitory activities in plants. We conducted a transcriptomic analysis to investigate how Ky-9 and Ky-72 enhance high-salinity stress tolerance. Although Ky-9 upregulated the expression of more genes than Ky-72, similar gene expression patterns were induced by both HDAC inhibitors. Additionally, the expression of high-salinity stress tolerance-related genes, such as anthocyanin-related genes and a small peptide-encoding gene, increased by Ky-9 and Ky-72. These data suggest that slight structural differences in chemical side chain between HDAC inhibitors can alter inhibitory effect on HDAC protein leading to influence gene expression, thereby enhancing high-salinity stress tolerance in different extent.
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http://dx.doi.org/10.1080/15592324.2018.1448333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5927655PMC
March 2018

The Distinct Roles of Class I and II RPD3-Like Histone Deacetylases in Salinity Stress Response.

Plant Physiol 2017 Dec 10;175(4):1760-1773. Epub 2017 Oct 10.

Plant Genomic Network Research Team, RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa 230-0045, Japan

Histone acetylation is an essential process in the epigenetic regulation of diverse biological processes, including environmental stress responses in plants. Previously, our research group identified a histone deacetylase (HDAC) inhibitor (HDI) that confers salt tolerance in Arabidopsis (). In this study, we demonstrate that class I HDAC (HDA19) and class II HDACs (HDA5/14/15/18) control responses to salt stress through different pathways. The screening of 12 different selective HDIs indicated that seven newly reported HDIs enhance salt tolerance. Genetic analysis, based on a pharmacological study, identified which HDACs function in salinity stress tolerance. In the wild-type Columbia-0 background, plants exhibit tolerance to high-salinity stress, while plants exhibit hypersensitivity to salt stress. Transcriptome analysis revealed that the effect of deficiency on the response to salinity stress is distinct from that of deficiencies. In plants, the expression levels of stress tolerance-related genes, late embryogenesis abundant proteins that prevent protein aggregation and positive regulators such as ABI5 and NAC019 in abscisic acid signaling, were induced strongly relative to the wild type. Neither of these elements was up-regulated in the plants. The mutagenesis of HDA19 by genome editing in the plants enhanced salt tolerance, suggesting that suppression of HDA19 masks the phenotype caused by the suppression of class II HDACs in the salinity stress response. Collectively, our results demonstrate that HDIs that inhibit class I HDACs allow the rescue of plants from salinity stress regardless of their selectivity, and they provide insight into the hierarchal regulation of environmental stress responses through HDAC isoforms.
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http://dx.doi.org/10.1104/pp.17.01332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717743PMC
December 2017

Live imaging of H3K9 acetylation in plant cells.

Sci Rep 2017 04 18;7:45894. Epub 2017 Apr 18.

Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.

Proper regulation of histone acetylation is important in development and cellular responses to environmental stimuli. However, the dynamics of histone acetylation at the single-cell level remains poorly understood. Here we established a transgenic plant cell line to track histone H3 lysine 9 acetylation (H3K9ac) with a modification-specific intracellular antibody (mintbody). The H3K9ac-specific mintbody fused to the enhanced green fluorescent protein (H3K9ac-mintbody-GFP) was introduced into tobacco BY-2 cells. We successfully demonstrated that H3K9ac-mintbody-GFP interacted with H3K9ac in vivo. The ratio of nuclear/cytoplasmic H3K9ac-mintbody-GFP detected in quantitative analysis reflected the endogenous H3K9ac levels. Under chemically induced hyperacetylation conditions with histone deacetylase inhibitors including trichostatin A, Ky-2 and Ky-14, significant enhancement of H3K9ac was detected by H3K9ac-mintbody-GFP dependent on the strength of inhibitors. Conversely, treatment with a histone acetyltransferase inhibitor, C646 caused a reduction in the nuclear to cytoplasmic ratio of H3K9ac-mintbody-GFP. Using this system, we assessed the environmental responses of H3K9ac and found that cold and salt stresses enhanced H3K9ac in tobacco BY-2 cells. In addition, a combination of H3K9ac-mintbody-GFP with 5-ethynyl-2'-deoxyuridine labelling confirmed that H3K9ac level is constant during interphase.
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http://dx.doi.org/10.1038/srep45894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5394682PMC
April 2017

Identification of Cyproheptadine as an Inhibitor of SET Domain Containing Lysine Methyltransferase 7/9 (Set7/9) That Regulates Estrogen-Dependent Transcription.

J Med Chem 2016 04 18;59(8):3650-60. Epub 2016 Apr 18.

Chemical Genetics Laboratory, RIKEN , 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

SET domain containing lysine methyltransferase 7/9 (Set7/9), a histone lysine methyltransferase (HMT), also methylates non-histone proteins including estrogen receptor (ER) α. ERα methylation by Set7/9 stabilizes ERα and activates its transcriptional activities, which are involved in the carcinogenesis of breast cancer. We identified cyproheptadine, a clinically approved antiallergy drug, as a Set7/9 inhibitor in a high-throughput screen using a fluorogenic substrate-based HMT assay. Kinetic and X-ray crystallographic analyses revealed that cyproheptadine binds in the substrate-binding pocket of Set7/9 and inhibits its enzymatic activity by competing with the methyl group acceptor. Treatment of human breast cancer cells (MCF7 cells) with cyproheptadine decreased the expression and transcriptional activity of ERα, thereby inhibiting estrogen-dependent cell growth. Our findings suggest that cyproheptadine can be repurposed for breast cancer treatment or used as a starting point for the discovery of an anti-hormone breast cancer drug through lead optimization.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01732DOI Listing
April 2016

Ky-2, a Histone Deacetylase Inhibitor, Enhances High-Salinity Stress Tolerance in Arabidopsis thaliana.

Plant Cell Physiol 2016 Apr 12;57(4):776-83. Epub 2015 Dec 12.

Plant Genomic Network Research Team, RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045 Japan Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama, Kanagawa, 244-0813 Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology (JST), 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012 Japan

Adaptation to environmental stress requires genome-wide changes in gene expression. Histone modifications are involved in gene regulation, but the role of histone modifications under environmental stress is not well understood. To reveal the relationship between histone modification and environmental stress, we assessed the effects of inhibitors of histone modification enzymes during salinity stress. Treatment with Ky-2, a histone deacetylase inhibitor, enhanced high-salinity stress tolerance in Arabidopsis. We confirmed that Ky-2 possessed inhibition activity towards histone deacetylases by immunoblot analysis. To investigate how Ky-2 improved salt stress tolerance, we performed transcriptome and metabolome analysis. These data showed that the expression of salt-responsive genes and salt stress-related metabolites were increased by Ky-2 treatment under salinity stress. A mutant deficient in AtSOS1(Arabidopis thaliana SALT OVERLY SENSITIVE 1), which encodes an Na(+)/H(+)antiporter and was among the up-regulated genes, lost the salinity stress tolerance conferred by Ky-2. We confirmed that acetylation of histone H4 at AtSOS1 was increased by Ky-2 treatment. Moreover, Ky-2 treatment decreased the intracellular Na(+)accumulation under salinity stress, suggesting that enhancement of SOS1-dependent Na(+)efflux contributes to increased high-salinity stress tolerance caused by Ky-2 treatment.
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http://dx.doi.org/10.1093/pcp/pcv199DOI Listing
April 2016

An efficient synthesis of SK-658 and its analogs as potent histone deacetylase inhibitors.

Bioorg Chem 2015 Apr 7;59:145-50. Epub 2015 Mar 7.

Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama 351-0198, Japan.

SK-658 is a potent histone deacetylase (HDAC) inhibitor that showed higher activity than SAHA due to the presence of extended hydrophobic group. We designed and synthesized thioester and SS-hybrid bearing SK-658 analogs as HDAC inhibitors. All the compounds were active in nano molar range and showed higher inhibitory activity than SAHA and SK-658. Among these, disulfide compounds showed the highest activity.
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http://dx.doi.org/10.1016/j.bioorg.2015.02.009DOI Listing
April 2015

Bicyclic tetrapeptide histone deacetylase inhibitors with methoxymethyl ketone and boronic acid zinc-binding groups.

Bioorg Chem 2014 Dec 22;57:121-126. Epub 2014 Oct 22.

Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama 351-0198, Japan.

Histone deacetylase (HDAC) inhibitors are a class of potential therapeutics for the treatment of cancer. Bicyclic tetrapeptides equipped with methoxymethyl ketone and boronic acid as zinc-binding group were designed and synthesized. The inhibitory activities of these compounds were evaluated against HDAC enzymes. The cell-free and cell-based assay data showed that both potency and selectivity changed with the change in zinc-binding group. Boronic acid-based compound showed poor activity whereas methoxymethyl ketone-based compound displayed impressive activity in both cell-free and cell-based conditions.
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http://dx.doi.org/10.1016/j.bioorg.2014.10.003DOI Listing
December 2014

Design and synthesis of mono and bicyclic tetrapeptides thioester as potent inhibitor of histone deacetylases.

Amino Acids 2014 Oct 22;46(10):2435-44. Epub 2014 Jul 22.

Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400, Serdang, Malaysia,

Inhibitors of histone deacetylases (HDACs) are a promising class of anticancer agents that have an effect on gene regulation. The naturally occurring cyclic depsipeptide FK228 containing disulfide and Largazole possessing thioester functionalities act as pro-drugs and share the same HDAC inhibition mechanism in cell. Inspired from these facts, we have reported bicyclic tetrapeptide disulfide HDAC inhibitors resembling FK228 with potent activity and enhanced selectivity. In the present study, we report the design and synthesis of several mono and bicyclic tetrapeptide thioester HDAC inhibitors that share the inhibition mechanism similar to Largazole. Most of the compounds showed HDAC1 and HDAC4 inhibition and p21 promoting activity in nanomolar ranges. Among these the monocyclic peptides 1, 2 and bicyclic peptide, 4 are notable demanding more advanced research to be promising anticancer drug candidates.
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http://dx.doi.org/10.1007/s00726-014-1800-5DOI Listing
October 2014

Bicyclic tetrapeptides as potent HDAC inhibitors: effect of aliphatic loop position and hydrophobicity on inhibitory activity.

Bioorg Med Chem 2014 Aug 26;22(15):3862-70. Epub 2014 Jun 26.

Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama 351-0198, Japan.

Several histone deacetylase (HDAC) inhibiting bicyclic tetrapeptides have been designed and synthesized through intramolecular ring-closing metathesis (RCM) reaction and peptide cyclization. We designed bicyclic tetrapeptides based on CHAP31, trapoxin B and HC-toxin I. The HDAC inhibitory and p21 promoter assay results showed that the aliphatic loop position as well as the hydrophobicity plays an important role toward the activity of the bicyclic tetrapeptide HDAC inhibitors.
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http://dx.doi.org/10.1016/j.bmc.2014.06.031DOI Listing
August 2014

Design and synthesis of CHAP31, trapoxin B and HC-toxin based bicyclic tetrapeptides disulfide as potent histone deacetylase inhibitors.

Bioorg Med Chem 2014 Aug 25;22(15):3850-5. Epub 2014 Jun 25.

Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama 351-0198, Japan.

The naturally occurring cyclic depsipeptide, FK228 inhibits histone deacetylase (HDAC) enzymes after reductive cleavage of intra-molecular disulfide bond. One of the sulfhydryl groups produced in the reduction interacts with zinc atom that involved in the catalytic mechanism of type 1 and 2 HDACs such as HDAC1, HDAC4, and HDAC6. In the present study, we describe the development of CHAP31, trapoxin B and HC-toxin based cyclic tetrapeptides with intra-molecular disulfide bond as HDAC inhibitors. The bicyclic tetrapeptides disulfide showed potent HDAC1 and HDAC4 inhibition and p21 promoting activity.
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http://dx.doi.org/10.1016/j.bmc.2014.06.029DOI Listing
August 2014

A convenient preparation of N (ε)-methyl-L-lysine derivatives and its application to the synthesis of histone tail peptides.

Amino Acids 2014 May 23;46(5):1305-11. Epub 2014 Feb 23.

Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Wakamatsu, Kitakyushu, 808-0196, Japan.

A convenient route is established for the preparation of N (α)-Fmoc-N (ε)-(Boc, methyl)-L-lysine and N (α)-Fmoc-N (ε)-dimethyl-L-lysine as building blocks to be used for the synthesis of methylated peptides. This methodology is based on the use of malonate derivatives and dibromobutane to produce key intermediates, L-2-amino-6-bromohexanoic acid derivatives, which could be modified to the required group at the ε-position. Fmoc-protection is accessible, so these compounds can be used in solution as well as in solid-phase peptide synthesis. Also the peptides containing these methylated lysines have been proved to resist the action of trypsin and lysyl endopeptidase. Thus, this new method could be considered as an improvement of the synthesis of N (ε)-methyl-L-lysine derivatives.
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http://dx.doi.org/10.1007/s00726-014-1690-6DOI Listing
May 2014

Design and synthesis of peptide-MCA substrates for a novel assay of histone methyltransferases and their inhibitors.

Bioorg Med Chem 2014 Feb 21;22(4):1268-75. Epub 2014 Jan 21.

Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, Saitama 351-0198, Japan.

Histone methyltransferases (HMTs) play an important role in controlling gene expression through site-specific methylation of lysines in core and linker histones within chromatin. As the typical HMTs, G9a and Set7/9 have been intensively studied that G9a is specific to the methylation at H3K9 and H3K27 and represses transcription, while Set7/9 methylates at H3K4. In this report we prepared various peptide-MCAs (4-methylcoumaryl-7-amides) related to histone tail and protein-substrates such as p53 and estrogen receptor-α. The fluorogenic substrates are applied for the assay of HMTs and an inhibitor, for example. The most sensitive and specific MCA-substrates to G9a and Set7/9 are discovered. The peptide-MCAs corresponding to the methylation sequences are promising for screening of HMT inhibitors.
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http://dx.doi.org/10.1016/j.bmc.2014.01.011DOI Listing
February 2014

Discovery of potent HDAC inhibitors based on chlamydocin with inhibitory effects on cell migration.

ChemMedChem 2014 Mar 27;9(3):627-37. Epub 2013 Nov 27.

School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, 116024 Dalian (China).

The histone deacetylase (HDAC) family is a promising drug target class owing to the importance of these enzymes in a variety of cellular processes. Docking studies were conducted to identify novel HDAC inhibitors. Subtle modifications in the recognition domain were introduced into a series of chlamydocin analogues, and the resulting scaffolds were combined with various zinc binding domains. Remarkably, cyclo(L-Asu(NHOH)-L-A3mc6c-L-Phe-D-Pro, compound 1 b), with a methyl group at positions 3 or 5 on the aliphatic ring, exhibited better antiproliferative effects than trichostatin A (TSA) against MCF-7 and K562 cell lines. In addition to cell-cycle arrest and apoptosis, cell migration inhibition was observed in cells treated with compound 1 b. Subsequent western blot analysis revealed that the balance between matrix metalloproteinase 2 (MMP2) and tissue inhibitors of metalloproteinase 1 (TIMP1) determines the degree of metalloproteinase activity in MCF-7 cells, thereby regulating cell migration. The improved inhibitory activity imparted by altering the hydrophobic substitution pattern at the bulky cap group is a valuable approach in the development of novel HDAC inhibitors.
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http://dx.doi.org/10.1002/cmdc.201300372DOI Listing
March 2014

Cyclic tetrapeptides with -SS- bridging between amino acid side chains for potent histone deacetylases' inhibition.

Amino Acids 2013 Oct 11;45(4):835-43. Epub 2013 Jun 11.

Department of Applied Chemistry, Kyushu Institute of Technology, Kitakyushu, 804-8550, Japan,

Cyclic depsipeptide FK228 with an intramolecular disulfide bond is a potent inhibitor of histone deacetylases (HDAC). FK228 is stable in blood because of its prodrug function, whose -SS- bond is reduced within the cell. Here, cyclic peptides with -SS- bridges between a variety of amino acids were synthesized and assayed for HDAC inhibition. Cyclic peptide 3, cyclo(-L-amino acid-L-amino acid-L-Val-D-Pro-), with an -SS- bridge between the first and second amino acids, was found to be a potent HDAC inhibitor. Cyclic peptide 7, cyclo(-L-amino acid-D-amino acid-L-Val-D-Pro-), with an -SS- bridge between the first and second amino acids, was also a potent HDAC inhibitor.
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http://dx.doi.org/10.1007/s00726-013-1527-8DOI Listing
October 2013

Dynamics and regulation of lysine-acetylation during one-cell stage mouse embryos.

Biochem Biophys Res Commun 2013 Apr 6;434(1):1-7. Epub 2013 Apr 6.

Division of Biological Science, Graduate School of Biology-Oriented Science and Technology, Kinki University, Kinokawa, Wakayama 649-6493, Japan.

Previous studies show that treatment of zygotes with trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), impacts the subsequent development to a blastocyst as well as full-term development. To reveal the dynamics of protein acetylation, with and without TSA treatment during one-cell stage, we examined oocytes and zygotes by immunofluorescence and Western Blot analyses using anti-acetylated lysine and acetylated α-tubulin antibodies. In unfertilized oocytes, lysine acetylation level was extremely low over all but faintly detected in the spindle. Once oocyte activation occurs, a dramatic increase of lysine acetylation signal was observed mostly in the pronuclei and a fiber-like structure, the so called midbody, suggesting activation coupled up-regulation of lysine acetylation presumably in histones and α-tubulin. TSA treatment resulted in significantly more hyperacetylation not only in the midbody structure and pronuclei but also in the whole cytoplasm. Consistently, Western Blot analysis revealed that acetylation of proteins about 53 kDa and 11 kDa in size, corresponding to α-tubulin and histone H4 sizes respectively, were increased mainly after oocyte activation and exclusively enhanced by TSA treatment in zygotes. To confirm this behavior of acetylated nonhistone proteins, acetylated α-tubulin was examined and found to be faintly detected in the spindle of MII oocytes but later in whole in the cell of zygotes including the midbody, which was enhanced by TSA treatment. To elucidate the mechanism underlying up-regulation of lysine acetylation following oocyte activation, we assayed the HDAC activity, and found significant reduction of HDAC activity from MII to zygotic stages. Taken together, our data indicate that HDACs play an important role in maintaining low acetylated status in a MII oocyte. However, once an oocyte has been activated, histone and nonhistone proteins including α-tubulin are hyperacetylated partly due to a reduction of HDAC activity. TSA treatment of zygotes enhances their acetylation, which could affect subsequent embryonic development.
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http://dx.doi.org/10.1016/j.bbrc.2013.03.083DOI Listing
April 2013

Mechanisms of G1 cell cycle arrest and apoptosis in myeloma cells induced by hybrid-compound histone deacetylase inhibitor.

Biochem Biophys Res Commun 2013 May 26;434(3):413-20. Epub 2013 Mar 26.

Division of Infections and Molecular Biology, Kyushu Dental University, Japan.

Objectives: Histone deacetylase (HDAC) inhibitors are new therapeutic agents, used to treat various types of malignant cancers. In the present study, we investigated the effects of Ky-2, a hybrid-compound HDAC inhibitor, on the growth of mouse myeloma cells.

Materials And Methods: Myeloma cells, HS-72, P3U1, and mouse normal cells were used in this study. Effect of HDAC inhibitors on cell viability was determined by WST-assay and trypan blue assay. Cell cycle was analyzed using flow cytometer. The expression of cell cycle regulatory and the apoptosis associated proteins were examined by Western blot analysis. Hoechst's staining was used to detect apoptotic cells.

Results: Our findings showed that Ky-2 decreased the levels of HDACs, while it enhanced acetylation of histone H3. Myeloma cell proliferation was inhibited by Ky-2 treatment. Interestingly, Ky-2 had no cytotoxic effects on mouse normal cells. Ky-2 treatment induced G1-phase cell cycle arrest and accumulation of a sub-G1 phase population, while Western blotting analysis revealed that expressions of the cell cycle-associated proteins were up-regulated. Also, Ky-2 enhanced the cleavage of caspase-9 and -3 in myeloma cells, followed by DNA fragmentation. In addition, Ky-2 was not found to induce apoptosis in bcl-2 overexpressing myeloma cells.

Conclusion: These findings suggest that Ky-2 induces apoptosis via a caspase-dependent cascade and Bcl-2-inhibitable mechanism in myeloma cells.
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http://dx.doi.org/10.1016/j.bbrc.2013.03.043DOI Listing
May 2013

Cyclic tetrapeptides with thioacetate tails or intramolecular disulfide bridge as potent inhibitors of histone deacetylases.

Bioorg Med Chem Lett 2012 Nov 8;22(21):6770-2. Epub 2012 Mar 8.

Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196, Japan.

Two thioacetate tails were introduced to the chlamydocin- and CHAP31-related cyclic tetrapeptides. An intramolecular disulfide bridge could be formed in the CHAP31-related cyclic peptides. Both the thioacetate-tailed and disulfide-bridged peptides were potent histone deacetylase inhibitors in the presence of sulfhydryl compound. Potent p21 promoter inducing activity was also observed in vivo.
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http://dx.doi.org/10.1016/j.bmcl.2012.03.004DOI Listing
November 2012

Identification of novel molecular targets regulated by tumor suppressive miR-375 induced by histone acetylation in esophageal squamous cell carcinoma.

Int J Oncol 2012 Sep 28;41(3):985-94. Epub 2012 Jun 28.

Department of Frontier Surgery, Chiba University Graduate School of Medicine, Chiba, Japan.

The aim of this study was to determine whether histone acetylation regulates tumor suppressive microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC) and to identify genes which are regulated by these miRNAs. We identified a miRNA that was highly upregulated in an ESCC cell line by cyclic hydroxamic acid-containing peptide 31 (CHAP31), one of the histone deacetylase inhibitors (HDACIs), using a miRNA array analysis. miR-375 was strongly upregulated by CHAP31 treatment in an ESCC cell line. The expression levels of the most upregulated miRNA, miR-375 were analyzed by quantitative real-time PCR in human ESCC specimens. The tumor suppressive function of miR-375 was revealed by restoration of miR-375 in ESCC cell lines. We performed a microarray analysis to identify target genes of miR-375. The mRNA and protein expression levels of these genes were verified in ESCC clinical specimens. LDHB and AEG-1/MTDH were detected as miR‑375-targeted genes. The restoration of miR-375 suppressed the expression of LDHB and AEG-1/MTDH. The ESCC clinical specimens exhibited a high level of LDHB expression at both the mRNA and protein levels. A loss-of-function assay using a siRNA analysis was performed to examine the oncogenic function of the gene. Knockdown of LDHB by RNAi showed a tumor suppressive function in the ESCC cells. The correlation between gene expression and clinicopathological features was investigated by immunohistochemistry for 94 cases of ESCC. The positive staining of LDHB correlated significantly with lymph node metastasis and tumor stage. It also had a tendency to be associated with a poor prognosis. Our results indicate that HDACIs upregulate miRNAs, at least some of which act as tumor suppressors. LDHB, which is regulated by the tumor suppressive miR-375, may therefore act as an oncogene in ESCC.
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http://dx.doi.org/10.3892/ijo.2012.1537DOI Listing
September 2012

Synthesis, evaluation and molecular modeling of cyclic tetrapeptide histone deacetylase inhibitors as anticancer agents.

J Pept Sci 2012 Apr 17;18(4):242-51. Epub 2012 Jan 17.

School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China.

Histone deacetylase inhibitors (HDACIs) are a promising class of anticancer agents. To examine whether a slight change in the recognition domain could alter their inhibitory activity, we synthesized a series of cyclo(-L-Am7(S2Py)-Aib-L-Phe(n-Me)-D-Pro)derivatives and evaluated their HDAC inhibitory and anticancer activities. The peptides exhibited potent HDAC inhibitory activity and inhibited three human cancer cell lines with IC₅₀ in the micromolar range. Docking and molecular dynamics simulation were conducted to explore the interaction mechanisms of class I and II HDACs with these inhibitors. It revealed that the zinc ion in the active site coordinated five atoms of HDACs and the sulfur atom of the inhibitor. The metal binding domains of these compounds interacted with HDAC2, and the surface recognition domains of these compounds interacted with HDAC4 through hydrogen bonding. The hydrophobic interactions also provided favorable contributions to stabilize the complexes. The results obtained from this study would be helpful for us to design some novel cyclic tetrapeptides that may act as potent HDACIs.
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http://dx.doi.org/10.1002/psc.2392DOI Listing
April 2012

Peptide-linked porphyrin sensitiser and colloidal Pt or Ir catalyst in the H2 formation reaction.

Photochem Photobiol Sci 2012 Feb 22;11(2):289-93. Epub 2011 Nov 22.

Faculty of Engineering, Kyushu Institute of Technology, Kitakyushu, 804-8550, Japan.

Porphyrins linking amphiphilic peptides were applied as photosensitisers, assuming that they would interact with the H(+) reduction catalyst, polymer-protected colloidal Pt or Ir. The close orientation of the porphyrin and metal catalyst may facilitate efficient electron transfer. The porphyrin linking a peptide containing glutamic acids (Glu), Ac-Cys(porph)-Glu-Val-Glu-Val-NH(2) (2), was an effective sensitiser for the H(2) generation reaction in the presence of N-benzyldihydronicotinamide and colloidal Pt or Ir in aqueous media under visible light illumination. At pH 5 and 7, 2 was a more efficient photosensitiser than tetrakis(p-carboxyphenyl)porphyrin (TCPP). The efficiency of H(+) reduction catalysts was in the order Ir-pGlu (polyglutamic acid) > Ir-PVP (polyvinylpyrrolidone) > Pt-PVP. Dynamic light scattering and scanning electron microscopy measurements showed that large particles formed when colloidal metal solutions were produced by microwave irradiation. Fluorescence quenching experiments suggested that electron transfer occurred from the photoexcited porphyrin to the colloidal Ir.
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http://dx.doi.org/10.1039/c1pp05250kDOI Listing
February 2012

Evaluation of functional groups on amino acids in cyclic tetrapeptides in histone deacetylase inhibition.

Amino Acids 2012 Jun 3;42(6):2103-10. Epub 2011 Jun 3.

Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Wakamatsu, Kitakyushu, 808-0196, Japan.

The naturally occurring cyclic tetrapeptide, chlamydocin, originally isolated from fungus Diheterospora chlamydosphoria, consists of α-aminoisobutyric acid, L-phenylalanine, D-proline and an unusual amino acid (S)-2-amino-8-((S)-oxiran-2-yl)-8-oxooctanoic acid (Aoe) and inhibits the histone deacetylases (HDACs), a class of regulatory enzymes. The epoxyketone moiety of Aoe is the key functional group for inhibition. The cyclic tetrapeptide scaffold is supposed to play important role for effective binding to the surface of enzymes. In place of the epoxyketone group, hydroxamic acid and sulfhydryl group have been applied to design inhibitor ligands to zinc atom in catalytic site of HDACs. In the research for more potent HDAC inhibitors, we replaced the epoxyketone moiety of Aoe with different functional groups and synthesized a series of chlamydocin analogs as HDAC inhibitors. Among the functional groups, methoxymethylketone moiety showed as potent inhibition as the hydroxamic acid. On the contrary, we confirmed that borate, trifruoromethylketone, and 2-aminoanilide are almost inactive in HDAC inhibition.
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http://dx.doi.org/10.1007/s00726-011-0947-6DOI Listing
June 2012

Real-time imaging of histone H4K12-specific acetylation determines the modes of action of histone deacetylase and bromodomain inhibitors.

Chem Biol 2011 Apr;18(4):495-507

Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan.

Histone acetylation constitutes an epigenetic mark for transcriptional regulation. Here we developed a fluorescent probe to visualize acetylation of histone H4 Lys12 (H4K12) in living cells using fluorescence resonance energy transfer (FRET) and the binding of the BRD2 bromodomain to acetylated H4K12. Using this probe designated as Histac-K12, we demonstrated that histone H4K12 acetylation is retained in mitosis and that some histone deacetylase (HDAC) inhibitors continue to inhibit cellular HDAC activity even after their removal from the culture. In addition, a small molecule that interferes with ability of the bromodomain to bind to acetylated H4K12 could be assessed using Histac-K12 in cells. Thus, Histac-K12 will serve as a powerful tool not only to understand the dynamics of H4K12-specific acetylation but also to characterize small molecules that modulate the acetylation or interaction status of histones.
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http://dx.doi.org/10.1016/j.chembiol.2011.02.009DOI Listing
April 2011

Histone deacetylase inhibitors block nuclear factor-κB-dependent transcription by interfering with RNA polymerase II recruitment.

Cancer Sci 2011 May 4;102(5):1081-7. Epub 2011 Mar 4.

Chemical Genetics Laboratory/Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama, Japan.

Histone deacetylase inhibitors (HDACi) have been shown to exhibit anti-inflammatory activity, but their mechanism of action is poorly understood. Trichostatin A (TSA) and the cyclic tetrapeptide class inhibitor Ky-2 inhibit both lipopolysaccharide-induced tumor necrosis factor-α (TNF-α) production in rats and TNF-α-induced expression of inflammatory genes in HeLa cells. We assessed the molecular mechanism underlying TSA-induced anti-inflammatory activity by genetically dissecting activation of the nuclear factor-κB (NF-κB) pathway following stimulation with TNF-α. Trichostatin A did not inhibit degradation of IκBα, nuclear translocation and DNA binding of NF-κB; also, the drug did not affect transient expression from exogenous κB-reporter plasmids. However, endogenous expression of inflammatory cytokines such as interleukin-8 (IL-8) was greatly reduced, even in the absence of de novo protein synthesis, suggesting that HDACi directly inhibits NF-κB-induced transcription. Indeed, chromatin immunoprecipitation (ChIP) analysis showed that events related to transcriptional activation of the IL-8 gene region in response to TNF-α, including recruitment of RNA polymerase II (Pol II), were compromised in the presence of TSA. These data indicate that HDAC activity is required for the efficient initiation and/or elongation of inflammatory gene transcription mediated by NF-κB.
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http://dx.doi.org/10.1111/j.1349-7006.2011.01904.xDOI Listing
May 2011

The role of class I histone deacetylase (HDAC) on gluconeogenesis in liver.

Biochem Biophys Res Commun 2011 Jan 23;404(1):166-72. Epub 2010 Nov 23.

Department of Metabolic Medicine, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.

Hepatic gluconeogenesis is crucial for glucose homeostasis. Although sirtuin 1 (Sirt1) is implicated in the regulation of gluconeogenesis in the liver, the effects of other histone deacetylases (HDAC) on gluconeogenesis are unclear. The aim of this study was to identify the role of class I HDACs in hepatic gluconeogenesis. In HepG2 cells and the liver of mice, the expressions of phosphoenol pyruvate carboxykinase (PEPCK) and hepatocyte nuclear factor 4α (HNF4α) were significantly decreased by treatment with a newly designed class I HDAC inhibitor, Ky-2. SiRNA knockdown of HDAC1 expression, but not of HDAC2 or HDAC3, in HepG2 cells decreased PEPCK and HNF4α expression. In HepG2 cells, insulin-stimulated phosphorylation of Akt and forkhead box O 1 (FoxO1) was increased by Ky-2. Pyruvate tolerance tests in Ky-2-treated high-fat-diet (HFD)-fed mice showed a marked reduction in blood glucose compared with vehicle-treated HFD mice. These data suggest that class I HDACs increase HNF4α protein expression and the transcriptional activity of FoxO1, followed by the induction of PEPCK mRNA expression and gluconeogenesis in liver.
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http://dx.doi.org/10.1016/j.bbrc.2010.11.086DOI Listing
January 2011

Efficient production of recombinant IgG by metabolic control and co-expression with GLUT5 in a fructose-based medium.

Cytotechnology 2010 Aug 10;62(4):301-6. Epub 2010 Aug 10.

The Cell Engineering Center, Kitakyushu National College of Technology, 5-20-1 Shii, Kokuraminami-ku, Kitakyushu-shi, Fukuoka, 802-0985, Japan,

A fructose-based cell culture is suitable for the process control of protein production because of slow sugar consumption rate and low lactate accumulation. The fructose transporter, GLUT5, mediates its incorporation into cells and is required for the fructose-based culture. In order to produce efficiently recombinant IgG by metabolic control and co-expression with GLUT5 in a fructose-based medium, an IgG and GLUT5 co-expression vector was constructed and transfected into the human myeloma derived cell line, SC-01MFP, which produced stably recombinant proteins. The cell proliferation in the fructose-based medium was improved by the GLUT5 gene transfection. The recombinant IgG production of the cells cultured in the fructose-based medium exhibited about two-fold increase of that in the glucose-based medium. Flow cytometoric analysis indicated that the GLUT5 protein expression level in cell surface was increased in the fructose-based medium. An exogenous but not endogenous GLUT5 transcription activator remarkably raised IgG productivity in the fructose-based medium when compared to that in the glucose-based medium, suggesting that exogenous GLUT5 expression may be involved in it. The GLUT5 co-expression system may be useful for efficient production of recombinant proteins by the fructose-based cell culture.
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http://dx.doi.org/10.1007/s10616-010-9289-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2978310PMC
August 2010

The role of the ribosomal protein S19 C-terminus in Gi protein-dependent alternative activation of p38 MAP kinase via the C5a receptor in HMC-1 cells.

Apoptosis 2010 Aug;15(8):966-81

Department of Molecular Pathology, Kumamoto University Graduate School, Japan.

We have demonstrated that an alternative C5a receptor (C5aR) ligand, the homodimer of ribosomal protein S19 (RP S19), contains a unique C-terminus (I(134)-H(145)) that is distinct from the moieties involved in the C5a-C5aR interaction. To examine the role of I(134)-H(145) in the ligand-C5aR interaction, we connected this peptide to the C-terminus of C5a (C5a/RP S19) and found that it endowed the second binding moiety of RP S19 (L(131)DR) with a relatively higher binding affinity to the C5aR on a human mast cell line, HMC-1. In contrast to the C5aR, the second C5aR C5L2 worked as a decoy receptor. As a result, the mitogen-activated protein kinase (MAPK) downstream of the Gi protein exchanged extracellular-signal regulated kinase for p38MAPK. This alternative p38MAPK activation could be pharmacologically suppressed not only by the downregulation of phosphoinositide 3-kinase (PI3K) by LY294002, but also by the over-activation of protein kinase C by phorbol 12-myristate 13-acetate. The activation was reproduced upon C5a-C5aR interaction by a simultaneous suppression of PI3K and phospholipase C with LY294002 and U73122 at low concentrations. Moreover, p38MAPK phosphorylation upstream of the pertussis toxin-dependent extracellular Ca(2+) entry was also suppressed by high concentrations of MgCl(2), which blocks melastatin-type transient receptor potential Ca(2+) channels (TRPMs). The active conformation of C5aR upon the ligation by C5a, at least on HMC-1 cells, is changed by the additional interaction of the I(134)-H(145) peptide, which seems to guide the alternative activation of p38MAPK. This activation is then amplified by a novel positive feedback loop between p38MAPK and TRPM.
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http://dx.doi.org/10.1007/s10495-010-0511-yDOI Listing
August 2010

CHAP31 induces apoptosis only via the intrinsic pathway in human esophageal cancer cells.

Oncology 2010 6;78(1):62-74. Epub 2010 Mar 6.

Department of Frontier Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. matsuhm @ faculty.chiba-u.jp

Objective: The antitumor mechanism of histone deacetylase (HDAC) inhibitors differs from conventional antitumor agents. HDAC inhibitors may be effective as novel therapeutic agents for esophageal squamous cell carcinoma (ESCC). This study describes the antiproliferative activity of CHAP31, a novel HDAC inhibitor. Furthermore, the molecular mechanism of CHAP31-induced apoptosis was investigated in ESCC.

Methods/results: The antitumor activity of CHAP31 was tested in esophageal cancer cell lines (T.Tn and TE2), and potent antitumor activity was observed in vitro and in vivo. In addition, CHAP31 induced apoptosis in esophageal cancer cells. Next, the mechanisms of CHAP31-induced apoptosis were examined using quantitative real-time RT-PCR and Western blotting. No processing of caspase 8 was observed, but CHAP31 induced the cleavage of caspase 9 and up-regulation of the Bax/Bcl-2 protein ratio.

Conclusion: This study provides new and important information on the potent antitumor activity of CHAP31 and the apoptotic pathway induced by CHAP31 in human esophageal cancer cell lines T.Tn and TE2. In contrast to previous reports showing that apoptosis induced by HDAC inhibitors includes the extrinsic pathway, in our study, apoptosis induced by CHAP31 in the human esophageal cell lines T.Tn and TE2 involved only the intrinsic pathway.
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http://dx.doi.org/10.1159/000292362DOI Listing
April 2010

Pivotal Advance: Interconversion between pure chemotactic ligands and chemoattractant/secretagogue ligands of neutrophil C5a receptor by a single amino acid substitution.

J Leukoc Biol 2010 Jun 20;87(6):965-75. Epub 2010 Jan 20.

Department of Molecular Pathology, Faculty of Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811, Japan.

Skp derived from Escherichia coli attracts leukocytes as a pure chemotactic ligand of the C5a receptor. We identified the submolecular region of Skp that binds and activates the C5a receptor to be -Gln103-Asp104-Arg105- using synthetic peptide fragments and site-directed mutants of Skp. As the C5a amino acid residue equivalent to Gln103 of Skp is Leu72, we prepared a Gln103Leu-Skp mutant as a recombinant protein. With this mutation, Skp gained secretagogue functions including induction of the respiratory burst and granule release reactions and leukotriene generation, in addition to the chemoattraction displayed by C5a. However, when we substituted Leu72 with Gln in C5a, the L72Q-C5a mutant largely lost its secretagogue function. These functional conversions were reproduced using synthetic peptides mimicking the receptor-binding/-activating regions of the recombinant proteins. Receptor-binding assays using the mimicking peptides demonstrated only a small difference between the Leu72-C5a and Gln72-C5a peptides. Consistently, L72Q-C5a apparently antagonized C5a secretagogue function. These results indicate that the difference between a chemotactic response and a combined chemotactic/secretory response can be attributed not to the nature of the receptor but to guidance by the ligand, at least in the case of C5a receptor-mediated leukocyte responses.
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http://dx.doi.org/10.1189/jlb.1009649DOI Listing
June 2010

Bicyclic peptides as potent inhibitors of histone deacetylases: optimization of alkyl loop length.

Bioorg Med Chem Lett 2010 Feb 21;20(3):997-9. Epub 2009 Dec 21.

Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196, Japan.

Bicyclic tetrapeptide hydroxamic acids were prepared as histone deacetylase (HDAC) inhibitors, and the evaluated inhibitory activity shows that they are potent against HDAC1 and HDAC4. The in vivo activity depends on alkyl loop length.
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http://dx.doi.org/10.1016/j.bmcl.2009.12.054DOI Listing
February 2010
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