論文 - 田村　悦臣

Methotrexate significantly induces apoptosis by inhibiting STAT3 activation in NPM-ALK-positive ALCL cells.

Uchihara Y, Komori R, Tago K, Tamura H, Funakoshi-Tago M

Biochemical pharmacology （Biochemical Pharmacology）  170   113666 2019年12月

研究論文（学術雑誌）,  ISSN  0006-2952

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© 2019 Elsevier Inc. Anaplastic large cell lymphoma (ALCL) is associated with a characteristic chromosomal translocation that generates the oncogenic fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). Methotrexate is a commonly used chemotherapeutic drug in the treatment of multiple cancers due to its inhibition of dihydrofolate reductase (DHFR), which suppresses the synthesis of DNA. In the present study, we found that low-dose methotrexate significantly induced apoptosis in transformed Ba/F3 cells expressing NPM-ALK by inhibiting the activation of signal transducer and activator of transcription factor 3 (STAT3), a critical downstream molecule of NPM-ALK. Although methotrexate prevented the phosphorylation of STAT3, it did not affect the activity of NPM-ALK. A co-treatment with folinic acid prevented the methotrexate-induced inhibition of STAT3 activation and induction of apoptosis, suggesting that methotrexate exerts its cytotoxic effects by depleting tetrahydrofolate (THF) in transformed cells by NPM-ALK. Furthermore, methotrexate induced the down-regulation of the anti-apoptotic protein, MCL-1, DNA damage, and the activation of a p53 tumor suppressor, leading to apoptosis through the inhibition of STAT3. Methotrexate significantly induced apoptosis in ALK inhibitor-resistant cells expressing the NPM-ALK mutant harboring the point mutation, G262R, and in ALCL patient-derived NPM-ALK-positive Ki-JK cells. Collectively, these results demonstrate the potential therapeutic application of methotrexate, which inhibits the activation of STAT3, to NPM-ALK-positive ALCL.

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Tyrosine-phosphorylated SOCS3 negatively regulates cellular transformation mediated by the myeloproliferative neoplasm-associated JAK2 V617F mutant

Funakoshi-Tago M., Tsuruya R., Ueda F., Ishihara A., Kasahara T., Tamura H., Tago K.

Cytokine （Cytokine）  123   154753 2019年11月

研究論文（学術雑誌）, 共著,  ISSN  10434666

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© 2019 Elsevier Ltd In the majority of myeloproliferative neoplasms (MPNs) patients, a point mutation, V617F has been found in Janus kinase 2 (JAK2) gene, and this JAK2 mutant provoked aberrant signaling pathway. In the current study, we found that suppressor of cytokine signaling proteins 3 (SOCS3) possessed the tumor suppressive activity against the JAK2 V617F mutant-provoked cellular transformation. The knockdown of SOCS3 increased the expression level of the JAK2 V617F mutant, which enhanced the activation of signaling mediators, including signal transducer and activator of transcription 3 and 5 (STAT3, STAT5) and extracellular signal-regulated kinase (ERK), and also increased of the proliferation rate and tumorigenesis activity of Ba/F3 cells expressing the JAK2 V617F mutant and erythropoietin receptor (EpoR). In contrast, the enforced expression of SOCS3 significantly inhibited the JAK2 V617F mutant-induced activation of downstream signaling molecules, cell proliferation, and tumorigenesis by down-regulating the expression level of the JAK2 V617F mutant. SOCS3 interacted with the JAK2V617F mutant through its SH2 domain and was phosphorylated at Tyr-204 and Tyr-221 in its SOCS box by the JAK2V617F mutant. SOCS3 mutants carrying a mutation in the SH2 domain (R71E) and a substitution at Tyr-221 (Y221F) failed to exert inhibitory effects on JAK2V617F mutant-induced cellular transformation and tumorigenesis. Collectively, these results imply that SOCS3 plays a negative role in the JAK2 V617F mutant-induced oncogenic signaling pathway through its SH2 domain and the phosphorylation of Tyr-221 in its SOCS box.

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3D in vivo imaging of the keratin filament network in the mouse stratum granulosum reveals profilaggrin-dependent regulation of keratin bundling.

Usui K, Kadono N, Furuichi Y, Shiraga K, Saitou T, Kawasaki H, Toyooka K, Tamura H, Kubo A, Amagai M, Matsui T

Journal of dermatological science （Journal of Dermatological Science）  94 （ 3 ） 346 - 349 2019年06月

研究論文（学術雑誌）, 共著,  ISSN  0923-1811

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N-Acetyl cysteine prevents activities of STAT3 inhibitors, Stattic and BP-1-102 independently of its antioxidant properties.

Uchihara Y, Ohe T, Mashino T, Kidokoro T, Tago K, Tamura H, Funakoshi-Tago M

Pharmacological reports : PR （Pharmacological Reports）  71 （ 6 ） 1067 - 1078 2019年05月

研究論文（学術雑誌）,  ISSN  1734-1140

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© 2019 Institute of Pharmacology, Polish Academy of Sciences Background: Inhibitors for signal transducer and activator of transcription 3 (STAT3), Stattic, BP-1-102, and LLL12 significantly induce apoptosis in transformed Ba/F3 cells expressing an oncogenic fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) that induces the activation of STAT3. We found that the antioxidant reagent, N-acetyl cysteine (NAC) prevented the abilities of Stattic and BP-1-102, but not LLL12 to induce apoptosis in transformed cells expressing NPM-ALK, providing a novel problem in use of STAT3 inhibitors. We herein investigated the mechanisms how NAC prevented the effects of Sttatic and BP-1-102. Methods: Ba/F3 cells expressing NPM-ALK and SUDHL-1 cells were treated with antioxidants such as NAC, Trolox or edaravone in combination with STAT3 inhibitors. Phosphorylation of STAT3, cell proliferation rate, cell viability, cell cycle, internucleosomal DNA fragmentation and the intracellular accumulation of reactive oxygen species (ROS) was investigated. The binding of STAT3 inhibitors and NAC was analyzed by LC–MS. Results: NAC but not Trolox and edaravone diminished the abilities of Stattic and BP-1-102 to induce apoptosis in cells expressing NPM-ALK. The ROS levels in cells expressing NPM-ALK were not markedly affected by the treatments with Stattic and BP-1-102 in combination with NAC, suggesting that NAC inhibited the activity of Stattic and BP-1-102 independent of its antioxidant activity. LC–MS analysis revealed that NAC directly bound to Stattic and BP-1-102. Furthermore, these NAC adducts exhibited no cytotoxicity, and failed to affect the activity of STAT3. Conclusions: NAC antagonizes the activities of Stattic and BP-1-102, which inhibit STAT3 activation by interacting with cysteine residues in STAT3.

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Coffee brew intake can prevent the reduction of lens glutathione and ascorbic acid levels in HFD-fed animals.

Nakazawa Y, Ishimori N, Oguchi J, Nagai N, Kimura M, Funakoshi-Tago M, Tamura H

Experimental and therapeutic medicine 17 （ 2 ） 1420 - 1425 2019年02月

研究論文（学術雑誌）,  ISSN  1792-0981

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Chemoenzymatic synthesis of hydroxytyrosol monoesters and their suppression effect on nitric oxide production stimulated by lipopolysaccharides

田村 悦臣

Bioscience, Biotechnology and Biochemistry （Bioscience, Biotechnology and Biochemistry）  83 （ 2 ） 185 - 191 2018年10月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  09168451

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© 2018 Japan Society for Bioscience, Biotechnology, and Agrochemistry. Fatty acid monoesters of hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol] were synthesized in two steps from tyrosol (4-hydroxyphenylethanol) by successive Candida antarctica lipase B-catalyzed chemoselective acylation on the primary aliphatic hydroxy group over phenolic hydroxy group in tyrosol, and 2-iodoxybenzoic acid (IBX)-mediated hydroxylation adjacent to the remaining free phenolic hydroxy group. Examination of their suppression effects on nitric oxide production stimulated by lipopolysaccharides in RAW264.7 cells showed that hydroxytyrosol butyrate exhibited the highest inhibition (IC 50 7.0 μM) among the tested compounds.

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Hydroxytyrosol butyrate inhibits 6-OHDA-induced apoptosis through activation of the Nrf2/HO-1 axis in SH-SY5Y cells

Funakohi-Tago M., Sakata T., Fujiwara S., Sakakura A., Sugai T., Tago K., Tamura H.

European Journal of Pharmacology （European Journal of Pharmacology）  834   246 - 256 2018年09月

研究論文（学術雑誌）, 共著,  ISSN  00142999

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© 2018 Elsevier B.V. Hydroxytyrosol (HT) is a polyphenol contained in olives and exhibits antioxidant activity. We herein investigated the effects of HT and its derivatives, hydroxytyrosol acetate (HT-A) and hydroxytyrosol butyrate (HT-B), on the protection of neuronal cells against apoptosis induced by the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA). The pretreatment of SH-SY5Y cells with HT-B, but not HT or HT-A significantly reduced the 6-OHDA-induced generation of reactive oxygen species, activation of caspase-3, and subsequent cell death. HT-B also induced the protein expression of the transcription factor, NF-E2-related factor-2 (Nrf2) and its transcriptional activation, resulting in the up-regulated expression of heme oxygenase-1 (HO-1), which conferred neuroprotection against 6-OHDA-induced oxidative damage. Furthermore, three cysteine residues, Cys151, Cys273, and Cys288 in Kelch-like ECH-associated protein 1 (Keap1) were necessary for the HT-B-induced activation of Nrf2. Collectively, the present results demonstrated that HT-B, harboring higher fat solubility than HT and HT-A, effectively elicited adaptive responses to oxidative stress by activating the Nrf2/HO-1 axis in neuronal cells.

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Roasted Coffee Reduces β-Amyloid Production by Increasing Proteasomal β-Secretase Degradation in Human Neuroblastoma SH-SY5Y Cells

田村 悦臣

Molecular Nutrition and Food Research （Molecular Nutrition and Food Research）  62 （ 21 ） e1800238 2018年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  16134125

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© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Scope: Epidemiological studies have shown that coffee consumption may be associated with a lower risk of developing several neurological disorders, including Alzheimer's disease (AD). Caffeine is a prominent candidate component underlying the preventive effects of coffee; however, the contribution of other constituents is unclear. To clarify this issue, the effect of roasting coffee beans on β-secretase (BACE1) expression in human neuroblastoma SH-SY5Y cells is investigated. Methods and results: Coffee (2%) reduces Aβ accumulation in culture medium to 80% of control levels after 24 h. Accordingly, BACE1 expression is decreased to 70% of control levels at 12 h. Experiments using cycloheximide and MG132, a proteasome inhibitor, reveal that coffee enhanced BACE1 degradation through activation of proteasomal activity. Furthermore, coffee activates cAMP-dependent protein kinase, and consequently, phosphorylation of a serine residue of proteasome 26S subunit, non-ATPase 11 (PSMD11). Pyrocatechol, a strong antioxidant known as catechol or 1,2-dihydroxybenzene, produced from chlorogenic acid during roasting, also reduces BACE1 expression by activation of proteasomal activity. Furthermore, pyrocatechol reduces Aβ production in SH-SY5Y cells. Conclusion: The data suggest that the roasting process may be crucial for the protective effects of coffee consumption in AD.

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Effect of hesperetin derivatives on the development of selenite-induced cataracts in rats

Nakazawa Y., Pauze M., Fukuyama K., Nagai N., Funakoshi-Tago M., Sugai T., Tamura H.

Molecular Medicine Reports （Molecular Medicine Reports）  18 （ 1 ） 1043 - 1050 2018年07月

研究論文（学術雑誌）, 共著,  ISSN  17912997

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Copyright © Spandidos Publications 2017. All rights reserved. Cataracts are a major cause of blindness worldwide. As anti-cataract pharmaceutical therapies require long-term treatment, identifying anti-cataract compounds that are ubiquitous in the human diet, have no adverse effects and are affordable, is of paramount importance. The present study focused on hesperetin and its derived compounds, hesperetin stearic acid ester (Hes-S) and hesperetin oleic acid ester (Hes-O), in order to investigate their therapeutic potential to treat cataracts in a selenite animal model. Thirteen-day-old Sprague Dawley rats were divided into 12 groups. Animals in groups 1 and 7 were subcutaneously injected with vehicle, those in groups 2 and 8 were administered hesperetin, those in groups 3 and 9 received stearic acid, those in groups 4 and 10 were injected with oleic acid, those in groups 5 and 11 were administered Hes-S, and those in groups 6 and 12 received Hes-O (10 nmol/kg body weight on days 0, 1 and 2). Animals in groups 7 to 12 were treated with sodium selenite (20 µmol/kg body weight given 4 h following the test compound treatment on day 0) to induce cataract. On day 6, rats had less severe central opacities and lower stage cataracts than rats in the selenite treatment-only control groups. The levels of glutathione (GSH) and ascorbic acid (AsA) in lenses with selenite-induced cataracts declined to one-third of that of controls, and the reduction in GSH and AsA levels was rescued following hesperetin, Hes-S or Hes-O treatment, with concentrations remaining to 70-80% of that of controls. However, there were no changes in the plasma levels of GSH and AsA following treatments. Administration of either hesperetin or hesperetin-derived compounds prevented the reduction of chaperone activity in the lens, and rats treated with Hes-S or Hes-O treatment had significantly greater chaperone activity than hesperetin-treated rats. Collectively, these results suggested that hesperetin and hesperetin-derived compounds may be novel drug compounds that have the potential to prevent or delay the onset of cataracts.

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A 5-hydroxyoxindole derivative attenuates LPS-induced inflammatory responses by activating the p38-Nrf2 signaling axis

田村 悦臣

Biochemical Pharmacology （Biochemical Pharmacology）  155   182 - 197 2018年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00062952

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© 2018 Elsevier Inc. 5-Hydroxyoxindole is a urinary metabolite of indole that exhibits antioxidant activity. In the present study, we found that a 5-hydroxyoxindole derivative (5-HI) significantly inhibited LPS-induced inflammatory effects in the murine macrophage cell line, RAW264.7. 5-HI induced the expression of the transcription factor, Nrf2, which is typically ubiquitinated by Keap1, an adaptor component of the ubiquitin E3 ligase complex, resulting in its proteasomal degradation. By utilizing Keap1−/− MEFs reconstituted with Keap1 mutants harboring substitutions in their major cysteine residues, we clarified the importance of Cys151 in Keap1 as a sensor for 5-HI in the induction of Nrf2 expression. Furthermore, 5-HI induced the activation of the MKK3/6-p38 pathway, which is required for the transcriptional activation of Nrf2. The knockdown of Nrf2 enhanced the LPS-induced expression of inflammatory mediators, including iNOS, NO, and CCL2, and effectively repressed the inhibitory effects of 5-HI on their expression. Although 5-HI and antioxidant N-acetyl cysteine (NAC) both reduced LPS-induced ROS generation, the treatment with NAC did not affect the LPS-induced expression of inflammatory mediators, suggesting that the anti-inflammatory activity of 5-HI mediated by Nrf2 is independent of redox control. Furthermore, when injected into mice with 5-HI, the expression of Nrf2 was significantly increased, and the LPS-induced mRNA expression of CXCL1, CCL2, TNFα and IL-6 were remarkably inhibited in the kidneys, liver, and lungs, and the production of these cytokines in serum was effectively reduced. Collectively, these results suggest that 5-HI has potential in the treatment of inflammatory diseases through the activation of Nrf2.

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Taxodione induces apoptosis in BCR-ABL-positive cells through ROS generation

田村 悦臣

Biochemical Pharmacology （Biochemical Pharmacology）  154   357 - 372 2018年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00062952

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© 2018 Elsevier Inc. Chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) are hematopoietic malignancies caused by the constitutive activation of BCR-ABL tyrosine kinase. Although direct BCR-ABL inhibitors, such as imatinib, were initially successful in the treatment of leukemia, many patients developed drug resistance over time due to the gatekeeper mutation of BCR-ABL T315I. In the present study, we found that taxodione, a quinone methide diterpene isolated from Taxodium distichum, significantly induced apoptosis in human myelogenous leukemia-derived K562 cells, which were transformed by BCR-ABL. Taxodione reduced the activities of mitochondrial respiratory chain (MRC) complexes III and V, which appeared to induce the production of reactive oxygen species (ROS). N-acetylcysteine (NAC), an antioxidant agent, canceled taxodione-induced ROS production, reductions in MRC activities, particularly complex V, and apoptotic cell death. Furthermore, in K562 cells treated with taxodione, BCR-ABL and its major signaling molecules, such as STAT5 and Akt were sequestered in mitochondrial fraction, and their localization changes decrease their abilities to stimulate cell proliferation, suggesting that these actions seem to be a mechanism how taxodione functions as an anti-tumor drug. Strikingly, NAC canceled these taxodione-caused anti-cancer effects. Taxodione induced apoptosis in transformed Ba/F3 cells induced not only by BCR-ABL, but also T315I-mutated BCR-ABL through the generation of ROS. Collectively, the present results suggest that in the treatment of leukemia, taxodione has potential as a compound with high efficacy to overcome BCR-ABL T315I mutation-mediated resistance in leukemia cells.

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Effect of hesperetin derivatives on the development of selenite‑induced cataracts in rats.

田村 悦臣

Molecular Medicine Reports 18   1043 - 1050 2018年03月

研究論文（学術雑誌）, 共著, 査読有り

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A major component of vitamin E, α-tocopherol inhibits the anti-tumor activity of crizotinib against cells transformed by EML4-ALK

田村 悦臣

European Journal of Pharmacology （European Journal of Pharmacology）  825   1 - 9 2018年02月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00142999

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© 2018 Elsevier B.V. Crizotinib is an inhibitor of anaplastic lymphoma kinase (ALK) and is of significant therapeutic benefit to patients with non-small cell lung cancer (NSCLC) harboring the EML4-ALK fusion gene. In the present study, we demonstrated that α-tocopherol, a major component of vitamin E, attenuated the effects of crizotinib independently of its anti-oxidant properties. α-Tocopherol significantly inhibited crizotinib-induced apoptosis in cells transformed by EML4-ALK. It also effectively attenuated the crizotinib-induced inhibition of EML4-ALK and its downstream molecules, STAT3 and ERK, and suppressed the inhibitory effects of crizotinib on EML4-ALK-mediated transformation in the focus formation assay. On the other hand, other members of the vitamin E family, namely, β-tocopherol, γ-tocopherol, δ-tocopherol, and α-tocotrienol, and a water-soluble analog of vitamin E, Trolox had no effects on the anti-tumor activity of crizotinib in cells transformed by EML4-ALK. Collectively, these results revealed the risk of the anti-tumor activity of crizotinib being attenuated when it is administrated in combination with vitamin E supplements containing α-tocopherol as a major component.

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Administration of antioxidant compounds affects the lens chaperone activity and prevents the onset of cataracts.

田村 悦臣

Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie （Biomedicine and Pharmacotherapy）  95   137 - 143 2017年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0753-3322

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© 2017 Elsevier Masson SAS To prevent lens opacification and cataract formation, the lens contains α-crystallin, which has been shown to function as a molecular chaperone that maintains the correct folding of other proteins. Oxidative stress is known to be an important factor in the initiation and progression of a cataract. So far, several antioxidant compounds have been reported to prevent cataracts in vivo and in vitro. This stress also triggers α-crystallin modifications and alters its chaperone activity. However, few studies have examined the relationship between the consumption of antioxidant compounds and lens chaperone activity. To elucidate the effect of antioxidants on lens chaperone activity, antioxidants were administered to a selenite-induced cataract model of rats. The chaperone activity in lens water-soluble fraction was measured using aldehyde dehydrogenase. All antioxidant treatment groups, except decaffeinated coffee treatment, had less severe central opacities and lower stage cataracts than control groups. The chaperone activity was weaker in lens of selenite cataract rats, but antioxidant compounds and coffee treatment can prevent the chaperone activity decreasing, but not decaffeinated coffee. These results suggested that the treatment with antioxidant compounds could prevent cataract formation by the maintenance of the chaperone activity in water-soluble lens proteins. Thus, this study describes the development of an anticataract drug target for lens chaperone activity.

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Occludin as a functional marker of vascular endothelial cells on tube-forming activity

田村 悦臣

Journal of Cellular Physiology （Journal of Cellular Physiology）  233 （ 2 ） 1700 - 1711 2017年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00219541

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© 2017 Wiley Periodicals, Inc. Cell therapy using endothelial progenitor cells (EPCs) is a promising strategy for the treatment of ischemic diseases. Two types of EPCs have been identified: early EPCs and late EPCs. Late EPCs are able to form tube structure by themselves, and have a high proliferative ability. The functional marker(s) of late EPCs, which relate to their therapeutic potential, have not been fully elucidated. Here we compared the gene expression profiles of several human cord blood derived late EPC lines which exhibit different tube formation activity, and we observed that the expression of occludin (OCLN) in these lines correlated with the tube formation ability, suggesting that OCLN is a candidate functional marker of late EPCs. When OCLN was knocked down by transfecting siRNA, the tube formation on Matrigel, the S phase + G 2 /M phase in the cell cycle, and the spheroid-based sprouting of late EPCs were markedly reduced, suggesting the critical role of OCLN in tube formation, sprouting, and proliferation. These results indicated that OCLN plays a novel role in neovascularization and angiogenesis.

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Alpha-tocopherol attenuates the anti-tumor activity of crizotinib against cells transformed by NPM-ALK.

田村 悦臣

PloS one （PLoS ONE）  12 （ 8 ） e0183003 2017年08月

研究論文（学術雑誌）, 共著, 査読有り

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© 2017 Uchihara et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Anaplastic large cell lymphomas (ALCL) are mainly characterized by harboring the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). The ALK inhibitor, crizotinib specifically induced apoptosis in Ba/F3 cells expressing NPM-ALK by inhibiting the activation of NPM-ALK and its downstream molecule, signal transducer and activator of transcription factor 3 (STAT3). We found that α-tocopherol, a major component of vitamin E, attenuated the effects of crizotinib independently of its anti-oxidant properties. Although α-tocopherol suppressed the inhibitory effects of crizotinib on the signaling axis including NPM-ALK and STAT3, it had no influence on the intake of crizotinib into cells. Crizotinib also directly inhibited the kinase activity of NPM-ALK; however, this inhibitory effect was not altered by the co-treatment with α-tocopherol. Whereas the nuclear localization of NPM-ALK was disappeared by the treatment with crizotinib, the co-treatment with α-tocopherol swept the effect of crizotinib and caused the localization of NPM-ALK in nucleus. The administration of α-tocopherol attenuated the anti-tumor activity of crizotinib against NPM-ALK-provoked tumorigenesis in vivo. Furthermore, the α-tocopherol-induced inhibition of crizotinib-caused apoptosis was also observed in NPM-ALK-positive cells derived from ALCL patients, namely, SUDHL-1 and Ki-JK. Collectively, these results not only revealed the novel mechanism underlying crizotinib-induced apoptosis in NPM-ALK-positive cells, but also suggest that the anti-tumor effects of crizotinib are attenuated when it is taken in combination with vitamin E.

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Coffee reduces KRAS expression in Caco-2 human colon carcinoma cells via regulation of miRNAs.

田村 悦臣

Oncology letters （Oncology Letters）  14 （ 1 ） 1109 - 1114 2017年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1792-1074

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© 2016, Spandidos Publications. All rights reserved. Previous epidemiological studies have demonstrated that moderate coffee consumption is associated with a lower risk of certain types of cancer, particularly colon cancer. To elucidate the molecular basis for this protective action, the effect of coffee on Caco-2 human colon carcinoma cells was investigated. Low concentrations of coffee (<5%) inhibited proliferation of Caco-2 cells without affecting cell viability. Coffee also reduced KRAS proto-oncogene, GTPase (KRAS) gene expression in a dose-dependent manner; however, caffeine, caffeic acid and chlorogenic acid, three major constituents of coffee, did not exhibit this effect. Increasing the duration of coffee bean roasting increased the reduction in KRAS expression, suggesting that the active constituents responsible for this effect emerged during the roasting process. MicroRNA (miR) analysis revealed that coffee induced the expression of miR-30c and miR-96, both of which target the KRAS gene. The results of the present study suggested that daily coffee consumption may reduce KRAS activity, thereby preventing the malignant growth of colon carcinoma cells.

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Coffee extract inhibits adipogenesis in 3T3-L1 preadipocyes by interrupting insulin signaling through the downregulation of IRS1.

田村 悦臣

PloS one （PLoS ONE）  12 （ 3 ） e0173264 2017年03月

研究論文（学術雑誌）, 共著, 査読有り

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© 2017 Maki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Although epidemiological data have indicated that a strong negative association exists between coffee consumption and the prevalence of obesity-associated diseases, the molecular mechanisms by which coffee intake prevents obesity-associated diseases has not yet been elucidated. In this study, we found that coffee intake significantly suppressed high-fat diet (HFD)-induced metabolic alternations such as increases in body weight and the accumulation of adipose tissue, and up-regulation of glucose, free fatty acid, total cholesterol and insulin levels in the blood. We also found that coffee extract significantly inhibited adipogenesis in 3T3-L1 preadipocytes. In the early phase of adipogenesis, 3T3-L1 cells treated with coffee extract displayed the retardation of cell cycle entry into the G2/M phase called as mitotic clonal expansion (MCE). Coffee extract also inhibited the activation of CCAAT/enhancer-binding protein β (C/EBPβ) by preventing its phosphorylation by ERK. Furthermore, the coffee extract suppressed the adipogenesis-related events such as MCE and C/EBPβ activation through the down-regulation of insulin receptor substrate 1 (IRS1). The stability of the IRS1 protein was markedly decreased by the treatment with coffee extract due to proteasomal degradation. These results have revealed an anti-adipogenic function for coffee intake and identified IRS1 as a novel target for coffee extract in adipogenesis.

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Data on the effect of hypomyelinating leukodystrophy 6 (HLD6)-associated mutations on the TUBB4A properties.

田村 悦臣

Data in brief 11   284 - 289 2017年02月

研究論文（学術雑誌）, 共著, 査読有り

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Roasting Enhances the Anti-Cataract Effect of Coffee Beans: Ameliorating Selenite-Induced Cataracts in Rats.

田村 悦臣

Current eye research （Current Eye Research）  42 （ 6 ） 864 - 870 2017年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0271-3683

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© 2017 Taylor & Francis. Purpose: Coffee is a widely consumed beverage. While recent studies have linked its intake to a reduced risk of cataracts, caffeine is believed to be the key factor for its effect. To know how roasting beans affects the effect of coffee on cataract formation, we investigated the impact roasting using a selenite-induced cataract rat model. Materials and Methods: Sprague Dawley rats were given a single injection of sodium selenite, which induced formation of nuclear cataracts by day 6, with or without coffee intake (100% coffee, 0.2 mL/day) for following 3 days. Results: The concentrations of glutathione (GSH) and ascorbic acid (AsA) in selenite-induced cataract lenses declined to half that of controls. However, 3 days of coffee intake ameliorated the reduction of GSH and AsA so that concentrations remained at 70–80% that of controls. Roasting enhanced the preventive effect of coffee by further reducing cataract formation and ameliorating selenite-induced reduction of antioxidants. High-performance liquid chromatography analysis revealed degradation of chlorogenic acid and generation of pyrocatechol during the coffee roasting process. We discovered that pyrocatechol, at doses equivalent to that found in dark-roasted coffee, was equally effective as caffeine at reducing cataract formation and ameliorating the reduction of antioxidants. Conclusion: Our results indicate that pyrocatechol, generated during the roasting process, acts as an antioxidant together with caffeine to prevent cataract formation.

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