Masahiko Ikekita

Publications (42)134.7 Total impact

• Article: SUTAF, a novel β-methoxyacrylate derivative, promotes neurite outgrowth with extracellular signal-regulated kinase and c-jun N-terminal kinase activation.

  Yukitoshi Nagahara, Eiji Suzuki, Yuriko Sekine, Hiromi Uchiro, Yoji Yoshimi, Takahisa Shinomiya, Masahiko Ikekita
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  ABSTRACT: β-Methoxyacrylate antibiotics are well known to inhibit the fungal and yeast mitochondrial respiratory chain. In addition, β-methoxyacrylates are reported to suppress the proliferation of mammalian cancer cells. Differentiation and cell-cycle arrest are closely related. The cell cycle of proliferating cells is suppressed before differentiation. In this study, we synthesized a β-methoxyacrylate analog and treated neuronal differential model cells with it. We then estimated β-methoxyacrylate's neurotrophic effect by inhibiting cell proliferation so as to orient neuronal differentiation. SUTAF-027-a novel β-methoxyacrylate derivative, arrested the cell cycle and thereby suppressed the proliferation of PC12 rat pheochromocytoma cells and mouse neuroblastoma Neuro2a cells at very low treatment doses, as low as 1nM. However, a single SUTAF-027 treatment did not affect neuritogenesis. Surprisingly, however, co-treatment of SUTAF-027 and nerve growth factor (NGF) significantly augmented the NGF-induced neurite outgrowth of PC12. On the other hand, a single treatment of 1nM SUTAF-027 induced neurite outgrowth in Neuro2a cells. Further signal transduction mechanism studies revealed that SUTAF-027 induced the phosphorylation of extracellular signal-regulated kinase (ERK) and slight phosphorylation of c-jun N-terminal kinase (JNK). Moreover, inhibition of ERK and JNK blocked SUTAF-027-augmented neurite outgrowth. These results suggested that the novel β-methoxyacrylate analog SUTAF-027 augmented neurite outgrowth by arresting the cell cycle and activating the ERK and JNK pathways.
  European journal of pharmacology 09/2012; 694(1-3):53-9. · 2.59 Impact Factor
• Article: Asn54-linked glycan is critical for functional folding of intercellular adhesion molecule-5.

  Tomohiro Ohgomori, Tomohisa Nanao, Akinori Morita, Masahiko Ikekita
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  ABSTRACT: Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendritically polarized type I membrane glycoprotein, and promotes dendritic filopodia formation. Although we have determined the N-glycan structures of ICAM-5 in a previous report, their function is unknown. Here, we produced fifteen ICAM-5 gene constructs, in which each potential N-glycosylation site was mutated, to elucidate the function of the N-glycans of ICAM-5, and observed the effects of transfection of them on a neuronal cell line, Neuro-2a (N2a). Only the N54Q mutant, which is the mutant for the most N-terminal glycosylation site, failed to induce filopodia-like protrusions in N2a cells. Immunofluorescence staining and cell surface biotinylation revealed that N54Q ICAM-5 was confined to the ER and also could not be expressed on the cell surface. This is further supported by the biochemical evidence that almost all N-glycans of N54Q ICAM-5 were digested by Endo glycosidase H and peptide:N-glycanase, indicating that almost all of them retain high-mannose-type structures in ER. In additon, it also failed to form disulfide bonds or functional protein complexes. The stable transformants of N54Q ICAM-5 showed retarded cell growth, but it was interesting that there was no apparent ER stress, because the mutant was sequentially degraded via ER associated degradation pathway by comparing the susceptibilities of the responses to various inhibitors of this pathway in wild-type and N54Q ICAM-5 transfectants. Taken together, the Asn(54)-linked glycan is necessary for normal trafficking and function of ICAM-5, but is unassociated with ER-associated degradation of it.
  Glycoconjugate Journal 12/2011; 29(1):47-55. · 2.12 Impact Factor
• Article: Inulin stimulates phagocytosis of PMA-treated THP-1 macrophages by involvement of PI3-kinases and MAP kinases.

  Yukitoshi Nagahara, Taome Nagamori, Hidekazu Tamegai, Mami Hitokuwada, Yoji Yoshimi, Masahiko Ikekita, Takahisa Shinomiya
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  ABSTRACT: Inulin is a polysaccharide that enhances various immune responses, mainly to T and B cells, natural killer cells, and macrophages in vivo and in vitro. Previous reports describe that inulin activates macrophages indirectly by affecting the alternative complement pathway. In this study, we examined the direct effect of inulin on PMA-treated THP-1 macrophages. Inulin treatment did not stimulate the proliferation of THP-1 macrophages at all. However, inulin treatment significantly increased phagocytosis of the polystyrene beads without the influence of serum. Doses of around 1 mg/mL had the maximal effect, and significant progression of phagocytosis occurred at times treated over 6 h. Inulin augmented phagocytosis not only with polystyrene beads but also with apoptotic cancer cells. The inulin-induced phagocytosis uptake was suppressed in Toll-like receptor (TLR) 4 mutated C3H/HeJ mice peritoneal macrophages. Moreover, inulin-induced THP-1 macrophage TNF-α secretion was inhibited using a blocking antibody specific to TLR4, suggesting that TLR4 is involved in the binding of inulin to macrophages. Furthermore, we used specific kinase inhibitors to assess the involvement of inulin-induced phagocytosis and revealed that phosphoinositide 3-kinase and mitogen-activated protein kinase, especially p38, participated in phagocytosis. These results suggest that inulin affects macrophages directly by involving the TLR4 signaling pathway and stimulating phagocytosis for enhancing immunomodulation.
  BioFactors 10/2011; 37(6):447-54. · 4.93 Impact Factor
• Article: 3-(3-Phenoxybenzyl)amino-β-carboline: a novel antitumor drug targeting α-tubulin.

  Reiko Ikeda, Masaki Kurosawa, Takazumi Okabayashi, Ayako Takei, Masamichi Yoshiwara, Tadashi Kumakura, Norio Sakai, Osamu Funatsu, Akinori Morita, Masahiko Ikekita, Yumi Nakaike, Takeo Konakahara
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  ABSTRACT: 3-(3-Phenoxybenzyl)amino-β-carboline 2h showed extremely-high activity; the IC(50) value was 0.074 μM. To verify 2h-induced cell death types, we observed the chromatin condensation, the DNA fragmentation and activated caspase-3 using Hoechst 33342, agarose electrophoresis and western blot, and suggesting 2h-induced cell death type was apoptosis. Flow cytometry showed that 2h-treated cell was induced SubG1 cell population after G2/M cell cycle arrest. In addition, using affinity chromatography and peptide mass fingerprinting, we found that interacting protein with this compound was α-tubulin protein.
  Bioorganic & medicinal chemistry letters 08/2011; 21(16):4784-7. · 2.65 Impact Factor
• Article: Transformation of thiols to disulfides by epolactaene and its derivatives.

  Kouji Kuramochi, Takashi Sunoki, Kazunori Tsubaki, Yoshiyuki Mizushina, Kengo Sakaguchi, Fumio Sugawara, Masahiko Ikekita, Susumu Kobayashi
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  ABSTRACT: In this paper we report a disulfide formation of thiols induced by epolactaene and its derivatives. We previously reported the disulfide formation of N-acetylcysteine methyl ester by epolactaene in a 1:1 MeOH/0.5M NaHCO(3) aq solution. The present studies reveal that the disulfide formation proceeds under mild conditions such as in PBS at pH 7.3, suggesting that epolactaene may induce disulfide formation of cellular thiols. This compound induces the disulfide formation of several thiols in a 1:1 MeOH/0.5M NaHCO(3) aq solution at room temperature. Moreover, our results show that the acyl side-chain of epolactaene greatly influences the products of the reaction. We analyzed the reaction mechanism by using thiolysis products of epolactaene derivatives and propose a new reaction mechanism.
  Bioorganic & medicinal chemistry 06/2011; 19(14):4162-72. · 2.82 Impact Factor
• Source

  Available from: Akinori Morita

  Article: Cycloheximide suppresses radiation-induced apoptosis in MOLT-4 cells with Arg72 variant of p53 through translational inhibition of p53 accumulation.

  Azusa Ito, Akinori Morita, Soichiro Ohya, Shinichi Yamamoto, Atsushi Enomoto, Masahiko Ikekita
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  ABSTRACT: The human T-cell leukemia cell line MOLT-4 is highly radiosensitive, and thus it is often used as a model of p53-dependent radiation-induced apoptosis. Two branches of the p53-mediated apoptotic pathway are reported: "transcription-dependent" and "transcription-independent." However, the relative contribution of each in different types of cells is not yet clearly defined. Moreover, recent studies have shown that the codon 72 polymorphic variants of p53 show different sensitivities to apoptosis signals. The Arg72 variant has a more potent apoptosis-inducing activity in mitochondria than the Pro72 variant. Here, we initially investigated the codon 72 polymorphism of p53 in MOLT-4 cells. Analysis of the p53 exon 4 genomic DNA sequence, which includes codon 72, revealed that MOLT-4 cells are homozygous for the allele encoding Arg72. We next investigated the involvement of the transcription-independent function of p53 using an RNA synthesis inhibitor, actinomycin D (ActD), and a protein synthesis inhibitor, cycloheximide (CHX), and found that the apoptosis was suppressed by CHX but not by ActD. We also revealed that the suppressive effect of CHX on apoptosis was specifically mediated by p53, using a p53-knockdown MOLT-4 transfectant. Furthermore, the suppressive effect of CHX on apoptosis was highly correlated with the suppression of p53 protein accumulation, and less correlated with the suppression of p53 target genes expression. These results indicated that p53 transactivation is not necessary to induce apoptosis, and that p53 protein accumulation itself is both necessary and sufficient to do so.
  Journal of Radiation Research 03/2011; 52(3):342-50. · 1.68 Impact Factor
• Source

  Available from: Akinori Morita

  Article: Ridaifen-G Induces Caspase-independent Atypical Cell Death

  Anniwaer Anlifeire, Manami Hatori, Akinori Morita, Isamu Shiina, Kenya Nakata, Yu-Ta Tosaki, Yan-Wen Wang, Masahiko Ikekita, Guan Li
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  ABSTRACT: We have investigated antitumor activities of Ridaifens (RIDs), which are a series of synthesized Tamoxifen (TAM) derivatives. In this study, we focused on one of RIDs, Ridaifen-G (RID-G), and investigated the cell death-inducing activity of it in four neoplastic hematopoietic cell lines, U937, Raji, THP-1, and IM-9 cells in the presence or absence of a pan-caspase inhibitor, Z-VAD-fmk. The aim of this study is to characterize the mode of RID-G-induced cell death, as compared with a typical apoptosis of etoposide-treated U937 cells, which die mainly through mitochondria-mediated apoptotic pathway in a caspase-dependent manner. To obtain reliable results, we analyzed RID-G-induced cell death by four methods, i.e., MTT assay, MitoTracker staining, AnnexinV-FITC/Propidium Iodide double staining, and DNA ladder formation. These analyses revealed that RID-G-induced cell death is accompanied by mitochondrial dysfunction and executed in a caspase-independent manner except DNA ladder formation. Z-VAD-fmk did not suppress the death, but suppressed etoposide-induced apoptosis in U937 cells. In DNA ladder formation analysis, RID-G induced a smear of DNA fragmentation in Raji and THP-1 cells, and RID-G-treated U937 cells showed less DNA ladder formation than when treated with etoposide. In addition, Z-VAD-fmk showed different effects on these DNA fragmentations: it largely suppressed, partialy suppressed, and on the contrary, enhanced DNA fragmentaion in U937, THP-1, and Raji cells, respectively. These results suggest that RID-G induces caspase-independent atypical cell death, which is accompanied by mitochondrial dysfunction.
  Chinese Journal of Cell Biology. 01/2011; 33:635–44.
• Article: Nobiletin, a citrus polymethoxyflavonoid, suppresses multiple angiogenesis-related endothelial cell functions and angiogenesis in vivo.

  Kazuhiro Kunimasa, Masahiko Ikekita, Mayumi Sato, Toshiro Ohta, Yukio Yamori, Megumi Ikeda, Sachi Kuranuki, Tsutomu Oikawa
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  ABSTRACT: Nobiletin is a citrus polymethoxyflavonoid that suppresses tumor growth and metastasis, both of which depend on angiogenesis. We recently identified nobiletin as a cell differentiation modulator. Because cell differentiation is a critical event in angiogenesis, it might be possible that nobiletin could exhibit antiangiogenic activity, resulting in suppression of these tumor malignant properties. To verify this possibility, we examined the antiangiogenic effects of nobiletin in vitro and in vivo. Nobiletin had concentration-dependent inhibitory effects on multiple functions of angiogenesis-related endothelial cells (EC); it suppressed the proliferation, migration and tube formation on matrigel of human umbilical vein EC (HUVEC) stimulated with endothelial cell growth supplement (ECGS), a mixture of acidic and basic fibroblast growth factors (FGFs). Gelatin zymography and northern blotting revealed that nobiletin suppressed pro-matrix metalloproteinase-2 (proMMP-2) production and MMP-2 mRNA expression in ECGS-stimulated HUVEC. Nobiletin also downregulated cell-associated plasminogen activator (PA) activity and urokinase-type PA mRNA expression. Furthermore, nobiletin inhibited angiogenic differentiation induced by vascular endothelial growth factor and FGF, an in vitro angiogenesis model. This inhibition was accompanied by downregulation of angiogenesis-related signaling molecules, such as extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase, and transcriptional factors (c-Jun and signal transducer and activator of transcription 3), and activation of the caspase pathway. In a chick embryo chorioallantoic membrane assay, nobiletin showed an antiangiogenic activity, the ID(50) value being 10μg (24.9nmol) per egg. These results indicate that nobiletin is a novel antiangiogenic compound that exhibits its activity through combined inhibition of multiple angiogenic EC functions.
  Cancer Science 11/2010; 101(11):2462-9. · 3.33 Impact Factor
• Article: Activin A induces neuronal differentiation and survival via ALK4 in a SMAD-independent manner in a subpopulation of human neuroblastomas.

  Kotaro Suzuki, Tomomi Kobayashi, Osamu Funatsu, Akinori Morita, Masahiko Ikekita
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  ABSTRACT: Activin A is a multifunctional homo-dimeric protein that belongs to the transforming growth factor (TGF)-beta superfamily. In neurons, activin has neuroprotective effects both in vitro and in vivo, but it inhibits neuronal differentiation in some cell lines. Here we report that activin A can promote neuronal differentiation in particular cases. We examined activin A-induced neuronal differentiation and survival in a selected subpopulation of a human neuroblastoma cell line, SK-N-SH, grown in low-serum (differentiation-inducing) conditions. Activin A caused dramatic neurite outgrowth, and increased the expression of neuronal markers and the transactivation of dopamine beta-hydroxylase. We demonstrated that the activin A signal is transduced through the activin A type 1 receptor, ALK4, and transactivates several TGF-beta target genes in a SMAD-independent manner. That is, activin A did not induce the phosphorylation of SMAD2/3, the interaction of SMAD2/3 with SMAD4, the binding of SMAD2/3 to the promoter of TGF-beta target genes, or the accumulation of SMAD2/3 in the nucleus. These results suggest that, in particular cases, activin A can induce neuronal differentiation and support neuronal survival in vitro. These findings may reflect previously unknown functions of activin A in neuronal cells in vivo.
  Biochemical and Biophysical Research Communications 03/2010; 394(3):639-45. · 2.48 Impact Factor
• Article: Design and synthesis of a fluorescent probe for Zn2+, 5,7-bis(N,N-dimethylaminosulfonyl)-8-hydroxyquinoline-pendant 1,4,7,10-tetraazacyclododecane and Zn2+-dependent hydrolytic and Zn2+-independent photochemical reactivation of its benzenesulfonyl-caged derivative.

  Ryosuke Ohshima, Masanori Kitamura, Akinori Morita, Motoo Shiro, Yasuyuki Yamada, Masahiko Ikekita, Eiichi Kimura, Shin Aoki
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  ABSTRACT: We previously reported on a 8-quinolinol-pendant cyclen (L(5)) as a Zn(2+) fluorophore (cyclen = 1,4,7,10-tetraazacyclododecane) and its caged derivative, 8-(benzenesulfonyloxy)-5-(N,N-dimethylaminosulfonyl)quinolin-2-ylmethyl-pendant cyclen (BS-caged-L(5)), which can be reactivated by hydrolysis of benzenesulfonyl group upon complexation with Zn(2+) at neutral pH to give a 1:1 Zn(2+)-L(5) complex (Zn(H(-1)L(5))). We report herein on the synthesis of 5,7-bis(N,N-dimethylaminosulfonyl)-8-hydroxyquinolin-2-ylmethyl-pendant cyclen (L(6)) and its caged derivative (BS-caged-L(6)) for more sensitive and more efficient cell-membrane permeability than those of L(5) and BS-caged-L(5). By potentiometric pH, (1)H NMR, and UV-vis spectroscopic titrations, the deprotonation constants pK(a1)-pK(a6) of H(5)L(6) were determined to be <2, <2, <2, 2.5 +/- 0.1 (for the 8-OH group of the quinoline moiety), 9.7 +/- 0.1, and 10.8 +/- 0.1 at 25 degrees C with I = 0.1 (NaNO(3)). The results of (1)H NMR, potentiometric pH, UV-vis, and fluorescent titrations showed that L(6) rapidly forms a 1:1 complex with Zn(2+) (Zn(H(-1)L(6))), the dissociation constant of which is 50 fM at pH 7.4. The fluorescent emission of Zn(H(-1)L(6)) at 478 nm is 32 times as large as that of L(6) (excitation at 370 nm), and the fluorescent quantum yield of Zn(H(-1)L(6)) (Phi(F) = 0.41) is much greater than that of Zn(H(-1)L(5)) (Phi(F) = 0.044). The BS-caged-L(6) was reactivated by hydrolysis of the benzenesulfonyl moiety more rapidly (completes in 30 min at pH 7.4 at 37 degrees C) than BS-caged-L(5), presumably enabling the practical detection of Zn(2+) in sample solutions and living cells. The photochemical deprotection of BS-caged-L(6) and the cell membrane permeability of L(6) and BS-caged-L(6) are also described.
  Inorganic Chemistry 02/2010; 49(3):888-99. · 4.60 Impact Factor
• Article: Sodium orthovanadate inhibits p53-mediated apoptosis.

  Akinori Morita, Shinichi Yamamoto, Bing Wang, Kaoru Tanaka, Norio Suzuki, Shin Aoki, Azusa Ito, Tomohisa Nanao, Soichiro Ohya, Minako Yoshino, Jin Zhu, Atsushi Enomoto, Yoshihisa Matsumoto, Osamu Funatsu, Yoshio Hosoi, Masahiko Ikekita
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  ABSTRACT: Sodium orthovanadate (vanadate) inhibits the DNA-binding activity of p53, but its precise effects on p53 function have not been examined. Here, we show that vanadate exerts a potent antiapoptotic activity through both transcription-dependent and transcription-independent mechanisms relative to other p53 inhibitors, including pifithrin (PFT) alpha. We compared the effects of vanadate to PFTalpha and PFTmicro, an inhibitor of transcription-independent apoptosis by p53. Vanadate suppressed p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. Similarly, vanadate suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of SaOS-2 cells. In radioprotection assays, which rely on p53, vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Together, our findings indicated that vanadate effectively suppresses p53-mediated apoptosis by both transcription-dependent and transcription-independent pathways, and suggested that both pathways must be inhibited to completely block p53-mediated apoptosis.
  Cancer Research 01/2010; 70(1):257-65. · 7.86 Impact Factor
• Article: Involvement of Galectin-3 with vascular cell adhesion molecule-1 in growth regulation of mouse BALB/3T3 cells.

  Tomomi Tadokoro, Masahiko Ikekita, Tosifusa Toda, Hiroko Ito, Takeshi Sato, Ryunosuke Nakatani, Yu Hamaguchi, Kiyoshi Furukawa
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  ABSTRACT: beta-Galactose residues on N-glycans have been implicated to be involved in growth regulation of cells. In the present study we compared the galactosylation of cell surface N-glycans of mouse Balb/3T3 cells between 30 and 100% densities and found the beta-1,4-galactosylation of N-glycans increases predominantly in a 100-kDa protein band on lectin blot analysis in combination with digestions by diplococcal beta-galactosidase and N-glycanase. When cells at 100% density were treated with jack bean beta-galactosidase, the incorporation of 5-bromodeoxyuridine into the cells was stimulated in a dose-dependent manner, suggesting the involvement of the galactose residues in growth regulation of cells. A galactose-binding protein was isolated from the plasma membranes of cells at 100% density by affinity chromatography using an asialo-transferrin-Sepharose column and found to be galectin-3 as revealed by mass spectrometric analysis. The addition of recombinant galectin-3 into cells at 50% density inhibited the incorporation of 5-bromodeoxyuridine in a dose-dependent manner, but the inhibition was prevented with haptenic sugar. An immunocytochemical study showed that galectin-3 is present at the surface of cells at 100% density but not at 30% density where it locates inside the cells. Several glycoproteins bind to a galectin-3-immobilized column, a major of which was identified as vascular cell adhesion molecule (VCAM)-1. Immunocytochemical studies showed that some galectin-3 and VCAM-1 co-localize at the surface of cells at 100% density, indicating that the binding of galectin-3 secreted from cells to VCAM-1 is one of the pathways involved in the growth regulation of Balb/3T3 cells.
  Journal of Biological Chemistry 11/2009; 284(51):35556-63. · 4.77 Impact Factor
• Article: Structural study of the N-glycans of intercellular adhesion molecule-5 (telencephalin).

  Tomohiro Ohgomori, Osamu Funatsu, Syu-ichi Nakaya, Akinori Morita, Masahiko Ikekita
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  ABSTRACT: Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendritically polarized membrane glycoprotein expressed in tissues distinct from those expressing other ICAMs. Here, we determined the N-glycan structure of ICAM-5 purified from adult rat brain and compared it with that of other ICAMs. N-glycans were released by N-glycosidase F digestion and labeled with p-amino benzoic octylester (ABOE). ABOE-labeled glycans were analyzed by high performance liquid chromatography (HPLC) and mass spectrometry. The N-glycans obtained from rat brain ICAM-5 consisted of approximately 85% neutral, 10.2% sialylated-only, 2.8% sulfated-only, and 1.2% sialylated and sulfated glycans. Compared with the N-glycan structures of human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells, rat brain ICAM-5 had less highly branched glycans, sialylated glycans, and N-acetyllactosamine structures. In contrast, high-mannose-type N-glycans and Lewis X were more commonly found in rat brain ICAM-5 than in human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells. In addition, sulfated glycans contained GlcNAc 6-O-sulfate on the non-reducing terminal side. Our data will be important for the elucidation of the roles of the N-glycans expressed in neural cells, including those present on ICAM-5.
  Biochimica et Biophysica Acta 10/2009; 1790(12):1611-23. · 4.66 Impact Factor
• Article: Uptake of a recombinant human alpha-L-iduronidase (laronidase) by cultured fibroblasts and osteoblasts.

  Takahiro Tsukimura, Youichi Tajima, Ikuo Kawashima, Tomoko Fukushige, Tamotsu Kanzaki, Takuro Kanekura, Masahiko Ikekita, Kanako Sugawara, Toshihiro Suzuki, Tadayasu Togawa, Hitoshi Sakuraba
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  ABSTRACT: To examine the uptake of a recombinant human alpha-L-iduronidase (laronidase) by cultured fibroblasts from a patient with mucopolysaccharidosis I (MPS I) and its effect on the cleavage of accumulated substrates, we performed enzymological, Western blotting, immunocytochemical and morphological studies. Laronidase was incorporated into the MPS I cells dose-dependently mainly via mannose 6-phosphate (M6P) receptors. Then the incorporated enzyme was transported to lysosomes and processed to the mature form, the pathological changes of the cells being improved. Furthermore, we compared the uptake of laronidase by cultured mouse osteoblasts with that by cultured mouse fibroblasts. The enzyme was incorporated into the cultured mouse osteoblasts mainly via M6P receptors, although mannose (Man) receptors were partially involved in the uptake of the enzyme, as in the cultured fibroblasts. But the uptake by the former was apparently lower than that by the latter. The administration of a high dose of the enzyme or development of a recombinant alpha-L-iduronidase containing many M6P residues is required for further improvement of enzyme replacement therapy for skeletal disorders caused by MPS I.
  Biological & Pharmaceutical Bulletin 10/2008; 31(9):1691-5. · 1.66 Impact Factor
• Article: Binding parameters and thermodynamics of the interaction of imino sugars with a recombinant human acid alpha-glucosidase (alglucosidase alfa): insight into the complex formation mechanism.

  Michiru Yoshimizu, Youichi Tajima, Fumiko Matsuzawa, Sei-Ichi Aikawa, Kunihiko Iwamoto, Toshihide Kobayashi, Tim Edmunds, Kaori Fujishima, Daisuke Tsuji, Kohji Itoh, Masahiko Ikekita, Ikuo Kawashima, Kanako Sugawara, Naho Ohyanagi, Toshihiro Suzuki, Tadayasu Togawa, Kazuki Ohno, Hitoshi Sakuraba
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  ABSTRACT: Recently, enzyme enhancement therapy (EET) for Pompe disease involving imino sugars, which act as potential inhibitors of acid alpha-glucosidases in vitro, to improve the stability and/or transportation of mutant acid alpha-glucosidases in cells was studied and attracted interest. However, the mechanism underlying the molecular interaction between the imino sugars and the enzyme has not been clarified yet. We examined the inhibitory and binding effects of four imino sugars on a recombinant human acid alpha-glucosidase, alglucosidase alfa, by means of inhibition assaying and isothermal titration calorimetry (ITC). Furthermore, we built structural models of complexes of the catalytic domain of the enzyme with the imino sugars bound to its active site by homology modeling, and examined the molecular interaction between them. All of the imino sugars examined exhibited a competitive inhibitory action against the enzyme, 1-deoxynojirimycin (DNJ) exhibiting the strongest action among them. ITC revealed that one compound molecule binds to one enzyme molecule and that DNJ most strongly binds to the enzyme among them. Structural analysis revealed that the active site of the enzyme is almost completely occupied by DNJ. These biochemical and structural analyses increased our understanding of the molecular interaction between a human acid alpha-glucosidase and imino sugars.
  Clinica Chimica Acta 06/2008; 391(1-2):68-73. · 2.54 Impact Factor
• Article: Syntheses and applications of fluorescent and biotinylated epolactaene derivatives: Epolactaene and its derivative induce disulfide formation.

  Kouji Kuramochi, Shunsuke Yukizawa, Seiki Ikeda, Takashi Sunoki, Satoshi Arai, Rie Matsui, Akinori Morita, Yoshiyuki Mizushina, Kengo Sakaguchi, Fumio Sugawara, Masahiko Ikekita, Susumu Kobayashi
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  ABSTRACT: Epolactaene, isolated from cultured Penicillium sp. BM 1689-P mycelium, induces neurite outgrowth and arrests the cell cycle of the human neuroblastoma cell line, SH-SY5Y, at the G1 phase. We have found that epolactaene and its derivatives induce apoptosis in the human leukemia B-cell line, BALL-1. In this study, we prepared fluorescent and biotinylated epolactaene derivatives. We characterized the cellular location and the identification of BALL-1 proteins that reacted with these compounds. The results obtained from the reaction of epolactaene or its derivative with N-acetylcysteine methyl ester indicate that these compounds induce the disulfide formation and the alpha-position of the epoxylactam core is the reactive site.
  Bioorganic & medicinal chemistry 06/2008; 16(9):5039-49. · 2.82 Impact Factor
• Article: BODIPY-based fluorescent redox potential sensors that utilize reversible redox properties of flavin.

  Yasuyuki Yamada, Yumiko Tomiyama, Akinori Morita, Masahiko Ikekita, Shin Aoki
  ChemBioChem 05/2008; 9(6):853-6. · 3.94 Impact Factor
• Article: Induction of mitochondria-involved apoptosis in estrogen receptor-negative cells by a novel tamoxifen derivative, ridaifen-B.

  Yukitoshi Nagahara, Isamu Shiina, Kenya Nakata, Akane Sasaki, Tomomi Miyamoto, Masahiko Ikekita
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  ABSTRACT: Tamoxifen is an antagonist of estrogen receptor, which is used widely as an estrogen receptor-positive breast cancer drug that blocks growth signals and provokes apoptosis. However, recent studies have revealed that tamoxifen induces apoptosis even in estrogen receptor-negative cells. In the present study, we synthesized several tamoxifen derivatives to augment the apoptosis-inducing effect of tamoxifen and evaluated the apoptosis-inducing pathway. The estrogen receptor-positive human leukemia cell line HL-60 and estrogen receptor-negative human leukemia cell line Jurkat were treated with tamoxifen and synthesized tamoxifen derivatives, and thereafter subjected to cell viability-detection assays. Tamoxifen derivatives, as well as the lead compound tamoxifen, decreased the cell viability despite the expression of estrogen receptor. Among all of the synthesized tamoxifen derivatives, ridaifen-B had more potent cancer cell-damaging activity than tamoxifen. Ridaifen-B fragmented Jurkat cell DNA and activated caspases, suggesting that the ridaifen-B-induced apoptosis pathway is estrogen receptor independent. Moreover, mitochondrial involvement during ridaifen-B-induced apoptosis was estimated. Ridaifen-B significantly reduced mitochondrial membrane potential, and overexpression of Bcl-2 inhibited ridaifen-B-induced apoptosis. These results suggest that the induction of apoptosis by ridaifen-B, a novel tamoxifen derivative, is dependent on mitochondrial perturbation without estrogen receptor involvement.
  Cancer Science 04/2008; 99(3):608-14. · 3.33 Impact Factor
• Article: Synthesis and pharmacological evaluation of the novel pseudo-symmetrical tamoxifen derivatives as anti-tumor agents.

  Isamu Shiina, Yoshiyuki Sano, Kenya Nakata, Takaaki Kikuchi, Akane Sasaki, Masahiko Ikekita, Yukitoshi Nagahara, Yoshimune Hasome, Takao Yamori, Kanami Yamazaki
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  ABSTRACT: Four pseudo-symmetrical tamoxifen derivatives, RID-B (13), RID-C (14), RID-D (15), and bis(dimethylaminophenetole) (16), were synthesized via the novel three-component coupling reaction, and the structure-activity relationships of these pseudo-symmetrical tamoxifen derivatives were examined. It was discovered that 13 and 16 strongly inhibit the viability of the HL-60 human acute promyelocytic leukemia cell line, whereas 14 possesses a medium activity against the same cell line and 15 has no effect on the cell viability. The global anti-tumor activity of 13-16 against a variety of human cancer cells was assessed using a panel of 39 human cancer cell lines (JFCR 39), and it was shown that RID-B (13) strongly inhibited the growth of several cancer cell lines at concentrations of less than 1 microM (at 0.38 microM for SF-539 [central nervous system], at 0.58 microM for HT-29 [colon], at 0.20 microM for DMS114 [lung], at 0.21 microM for LOX-IMVI [melanoma], and at 0.23 microM for MKN74 [stomach]).
  Biochemical pharmacology 04/2008; 75(5):1014-26. · 4.25 Impact Factor
• Article: An expeditious synthesis of tamoxifen, a representative SERM (selective estrogen receptor modulator), via the three-component coupling reaction among aromatic aldehyde, cinnamyltrimethylsilane, and beta-chlorophenetole.

  Isamu Shiina, Yoshiyuki Sano, Kenya Nakata, Masahiko Suzuki, Toshikazu Yokoyama, Akane Sasaki, Tomoko Orikasa, Tomomi Miyamoto, Masahiko Ikekita, Yukitoshi Nagahara, Yoshimune Hasome
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  ABSTRACT: Two new synthetic pathways to the anti-cancer agent tamoxifen and its derivatives were developed. The first route involved the aldol reaction of benzyl phenyl ketone with acetaldehyde followed by Friedel-Crafts substitution with anisole in the presence of Cl(2)Si(OTf)(2) to produce 1,1,2-triaryl-3-acetoxybutane, a precursor of the tamoxifen derivatives. The second one utilized the novel three-component coupling reaction among aromatic aldehydes, cinnamyltrimethylsilane, and aromatic nucleophiles using HfCl(4) as a Lewis acid catalyst to produce 3,4,4-triarylbutene, that is also a valuable intermediate of the tamoxifen derivatives. The former strategy requires a total of 10 steps from the aldol formation to the final conversion to tamoxifen, whereas the latter needs only three or four steps to produce tamoxifen and droloxifene including the installation of the side-chain moiety and the base-induced double-bond migration to form the tetra-substituted olefin structure. This synthetic strategy seems to serve as a new and practical pathway to prepare not only the tamoxifen derivatives but also the other SERMs (selective estrogen receptor modulators) including estrogen-dependent breast cancer and osteoporosis agents.
  Bioorganic & medicinal chemistry 01/2008; 15(24):7599-617. · 2.82 Impact Factor