論文 - 多胡　めぐみ

Oral consumption of α-glucosyl-hesperidin could prevent lens hardening, which causes presbyopia

Nakazawa Y., Aoki M., Doki Y., Morishita N., Endo S., Nagai N., Funakoshi-Tago M., Tamura H.

Biochemistry and Biophysics Reports （Biochemistry and Biophysics Reports）  25 2021年03月

研究論文（学術雑誌）, 共著, 査読有り

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© 2020 The Authors Presbyopia is one of the most well-known diseases of the eye, predominantly affecting the adult population after 50 years’. Due to hardening of the lens and failure of accommodative change, patients lose the ability to focus on near objects. This eye symptom is reported to be an early symptom of age-related nuclear cataract, and we have previously reported that hesperetin treatment could delay the onset of nuclear cataractogenesis induced by sodium selenite. In this study, we examined whether oral intake of α-glucosyl-hesperidin (G-Hsd), which has greater water solubility than hesperetin, could delay the onset of presbyopia. G-Hsd treatment protected lens elasticity, upregulated the mRNA expression of anti-oxidative enzymes like glutathione reductase and thioredoxin reductase 1 in the plasma and lens, and prevented premature cataract symptoms in selenite-induced cataract rat lens. Thus, the anti-presbyopic effects of G-Hsd were attributed, at least in part, to its antioxidant effects. G-Hsd represents the first oral treatment agent with anti-presbyopia and/or anti-cataract properties.

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Effect of alpha‐glucosyl‐hesperidin consumption on lens sclerosis and presbyopia

Nakazawa Y., Doki Y., Sugiyama Y., Kobayashi R., Nagai N., Morisita N., Endo S., Funakoshi‐tago M., Tamura H.

Cells （Cells）  10 （ 2 ） 1 - 11 2021年02月

研究論文（学術雑誌）, 共著, 査読有り

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Presbyopia is characterized by a decline in the ability to accommodate the lens. The most commonly accepted theory for the onset of presbyopia is an age‐related increase in the stiffness of the lens. However, the cause of lens sclerosis remains unclear. With age, water microcirculation in the lens could change because of an increase in intracellular pressure. In the lens, the intracellular pressure is controlled by the Transient Receptor Potential Vanilloid (TRPV) 1 and TRPV4 feedback pathways. In this study, we tried to elucidate that administration of α‐glucosyl‐hesperidin (G‐Hsd), previously reported to prevent nuclear cataract formation, affects lens elasticity and the distribution of TRPV channels and Aquaporin (AQP) channels to meet the requirement of intracellular pressure. As a result, the mouse control lens was significantly toughened compared to both the 1% and 2% G‐Hsd mouse lens treatments. The anti‐oxidant levels in the lens and plasma decreased with age; however, this decrease could be nullified with either 1% or 2% G‐Hsd treatment in a concentration-and exposure time‐dependent manner. Moreover, G‐Hsd treatment affected the TRPV4 distribu-tion, but not TRPV1, AQP0, and AQP5, in the peripheral area and could maintain intracellular pres-sure. These findings suggest that G‐Hsd has great potential as a compound to prevent presbyopia and/or cataract formation.

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EBP2, a novel NPM-ALK-interacting protein in the nucleolus, contributes to the proliferation of ALCL cells by regulating tumor suppressor p53

Uchihara Y., Tago K., Tamura H., Funakoshi-Tago M.

Molecular Oncology （Molecular Oncology）  15 （ 1 ） 167 - 197 2021年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  15747891

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The oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), found in anaplastic large-cell lymphoma (ALCL), localizes to the cytosol, nucleoplasm, and nucleolus. However, the relationship between its localization and transforming activity remains unclear. We herein demonstrated that NPM-ALK localized to the nucleolus by binding to nucleophosmin 1 (NPM1), a nucleolar protein that exhibits shuttling activity between the nucleolus and cytoplasm, in a manner that was dependent on its kinase activity. In the nucleolus, NPM-ALK interacted with Epstein–Barr virus nuclear antigen 1-binding protein 2 (EBP2), which is involved in rRNA biosynthesis. Moreover, enforced expression of NPM-ALK induced tyrosine phosphorylation of EBP2. Knockdown of EBP2 promoted the activation of the tumor suppressor p53, leading to G0/G1-phase cell cycle arrest in Ba/F3 cells transformed by NPM-ALK and ALCL patient-derived Ki-JK cells, but not ALCL patient-derived SUDH-L1 cells harboring p53 gene mutation. In Ba/F3 cells transformed by NPM-ALK and Ki-JK cells, p53 activation induced by knockdown of EBP2 was significantly inhibited by Akt inhibitor GDC-0068, mTORC1 inhibitor rapamycin, and knockdown of Raptor, an essential component of mTORC1. These results suggest that the knockdown of EBP2 triggered p53 activation through the Akt-mTORC1 pathway in NPM-ALK-positive cells. Collectively, the present results revealed the critical repressive mechanism of p53 activity by EBP2 and provide a novel therapeutic strategy for the treatment of ALCL.

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The role of MerTK in promoting cell migration is enhanced by the oncogenic Ras/IL-33 signaling axis

Ohta S., Tago K., Kuchimaru T., Funakoshi-Tago M., Horie H., Aoki-Ohmura C., Matsugi J., Yanagisawa K.

FEBS Journal （FEBS Journal）  289 （ 7 ） 1950 - 1967 2021年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1742464X

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Ras genes are frequently mutated in many cancer types; however, there are currently no conclusively effective anticancer drugs against Ras-induced cancer. Therefore, the downstream effectors of Ras signaling need to be identified for the development of promising novel therapeutic approaches. We previously reported that oncogenic Ras induced the expression of NF-HEV/IL-33, a member of the interleukin-1 family, and showed that intracellular IL-33 was required for oncogenic Ras-induced cellular transformation. In the present study, we demonstrated that the c-Mer proto-oncogene tyrosine kinase (MerTK), a receptor tyrosine kinase, played essential roles in oncogenic Ras/IL-33 signaling. The expression of MerTK was enhanced in transformed NIH-3T3 cells by the expression of oncogenic Ras, H-Ras (G12V), in an IL-33-dependent manner. In human colorectal cancer tissues, MerTK expression also correlated with IL-33 expression. The knockdown of IL-33 or MerTK effectively attenuated the migration of NIH-3T3 cells transformed by H-Ras (G12V) and A549, LoVo, and HCT116 cells harboring an oncogenic K-Ras mutation. Furthermore, the suppression of Ras-induced cell migration by the knockdown of IL-33 was rescued by the enforced expression of MerTK. The present results also revealed that MerTK was effectively phosphorylated in NIH-3T3 cells transformed by Ras (G12V). Ras signaling was essential for the tyrosine phosphorylation of MerTK, and the kinase activity of MerTK was indispensable for accelerating cell migration. Collectively, the present results reveal a novel role for MerTK in cancer malignancy, which may be utilized to develop novel therapeutic strategies that target Ras-transformed cells.

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Pyrocatechol, a component of coffee, suppresses LPS-induced inflammatory responses by inhibiting NF-κB and activating Nrf2

Funakoshi-Tago M., Nonaka Y., Tago K., Takeda M., Ishihara Y., Sakai A., Matsutaka M., Kobata K., Tamura H.

Scientific Reports （Scientific Reports）  10 （ 1 ）  2020年12月

研究論文（学術雑誌）, 共著, 査読有り

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© 2020, The Author(s). Coffee is a complex mixture of many bioactive compounds possessing anti-inflammatory properties. However, the mechanisms by which coffee exerts anti-inflammatory effects remains unclear and the active ingredients have not yet been identified. In this study, we found that coffee extract at more than 2.5%(v/v) significantly inhibited LPS-induced inflammatory responses in RAW264.7 cells and that anti-inflammatory activity of coffee required the roasting process. Interestingly, we identified pyrocatechol, a degradation product derived from chlorogenic acid during roasting, as the active ingredient exhibiting anti-inflammatory activity in coffee. HPLC analysis showed that 124 μM pyrocatechol was included in 100% (v/v) roasted coffee. A treatment with 5%(v/v) coffee extract and more than 2.5 μM pyrocatechol inhibited the LPS-induced activation of NF-κB and also significantly activated Nrf2, which acts as a negative regulator in LPS-induced inflammation. Furthermore, intake of 60% (v/v) coffee extract and 74.4 μM pyrocatechol, which is the concentration equal to contained in 60% (v/v) coffee, markedly inhibited the LPS-induced inflammatory responses in mice. Collectively, these results demonstrated that pyrocatechol, which was formed by the roasting of coffee green beans, is one of the ingredients contributing to the anti-inflammatory activity of coffee.

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Coffee decoction enhances tamoxifen proapoptotic activity on MCF-7 cells

Funakoshi-Tago M., Tago K., Li C., Hokimoto S., Tamura H.

Scientific Reports （Scientific Reports）  10 （ 1 ） 19588 2020年11月

研究論文（学術雑誌）, 共著, 査読有り

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The consumption of coffee has been suggested to effectively enhance the therapeutic effects of tamoxifen against breast cancer; however, the underlying molecular mechanisms remain unclear. We herein attempted to clarify how coffee decoction exerts anti-cancer effects in cooperation with tamoxifen using the estrogen receptor α (ERα)-positive breast cancer cell line, MCF-7. The results obtained showed that coffee decoction down-regulated the expression of ERα, which was attributed to caffeine inhibiting its transcription. Coffee decoction cooperated with tamoxifen to induce cell-cycle arrest and apoptotic cell death, which may have been mediated by decreases in cyclin D1 expression and the activation of p53 tumor suppressor. The inclusion of caffeine in coffee decoction was essential, but not sufficient, to induce cell-cycle arrest and apoptotic cell death, suggesting the requirement of unknown compound(s) in coffee decoction to decrease cyclin D1 expression and activate apoptotic signaling cascades including p53. The activation of p53 through the cooperative effects of these unidentified component(s), caffeine, and tamoxifen appeared to be due to the suppression of the ERK and Akt pathways. Although the mechanisms by which the suppression of these pathways induces p53-mediated apoptotic cell death remain unclear, the combination of decaffeinated coffee, caffeine, and tamoxifen also caused cell-cycle arrest and apoptotic cell death, suggesting that unknown compound(s) present in decaffeinated coffee cooperate with caffeine and tamoxifen.

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Kampo medicines, Rokumigan, Hachimijiogan, and Goshajinkigan, significantly inhibit glucagon-induced CREB activation

Funakoshi-Tago M., Yu S., Kushida A., Takeuchi K., Tamura H.

Heliyon （Heliyon）  6 （ 3 ）  2020年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  24058440

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© 2020 The Author(s) The pathophysiology of type 2 diabetes mellitus (T2DM) is characterized by not only insulin resistance, but also the abnormal regulation of glucagon secretion, suggesting that antagonizing the glucagon-induced signaling pathway has therapeutic potential in the treatment of T2DM. Although various Kampo medicines (traditional herbal medicines) are often utilized to ameliorate the symptoms of T2DM, their effects on glucagon signaling have not yet been clarified. In the present study, we examined the effects of nine types of representative Kampo formulations prescribed for T2DM on glucagon-induced CREB activation in HEK293T cells stably expressing glucagon receptor (Gcgr) and a hepatic cell line HepG2. Among these Kampo medicines, Rokumigan, Hachimijiogan, and Goshajinkigan significantly suppressed the glucagon-induced transactivation of the cAMP-responsive element (CRE)-binding protein (CREB) by inhibiting its interaction with CREB-binding protein (CBP), which led to a reduction in the expression of phosphoenolpyruvate carboxykinase (PEPCK) mRNA. Furthermore, among the crude drugs commonly contained in these three Kampo medicines, Rehmannia Root (Jio), Moutan Bark (Botampi), and Cornus Fruit (Shanzhuyu) exerted inhibitory effects on glucagon-induced CREB activation. Collectively, the present results provide a novel mechanism, the inhibition of glucagon signaling, by which Rokumigan, Hachimijiogan, and Goshajinkigan improve the symptoms of T2DM.

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Oral intake of α-glucosyl-hesperidin ameliorates selenite-induced cataract formation

Nakazawa Y., Aoki M., Ishiwa S., Morishita N., Endo S., Nagai N., Yamamoto N., Funakoshi-Tago M., Tamura H.

Molecular Medicine Reports （Molecular Medicine Reports）  21 （ 3 ） 1258 - 1266 2020年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  17912997

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© 2020 Spandidos Publications. All rights reserved. Hesperetin is a natural flavonoid with robust antioxidant properties. Our previous study reported that hesperetin can prevent cataract formation. However, an important consideration regarding hesperetin consumption is the limited bioavailability due to its poor solubility. The present study investigated the anti-cataract effects of α-glucosyl hesperidin in vivo and in vitro using a selenite-induced cataract model. SD rats (age, 13 days) were orally administered PBS (0.2 ml) or α-glucosyl hesperidin (200 mg/kg) on days 0, 1 and 2. Sodium selenite was subcutaneously administered to the rats 4 h after the first oral administration on day 0. Antioxidant levels in the lens and blood were measured on day 6. In vitro, human lens epithelial cells were treated with sodium selenite (10 μM) and/or hesperetin (50 or 100 mM) for 24 h and analyzed for apoptosis markers using sub-G1 population and Annexin V-FITC/propidium iodide staining and DNA ladder formation. α-glucosyl hesperidin treatment significantly reduced the severity of selenite-induced cataract. The level of antioxidants was significantly reduced in the selenite-treated rats compared with in the controls; however, they were normalized with α-glucosyl hesperidin treatment. In vitro, hesperetin could significantly reduce the number of cells undergoing apoptosis induced by sodium selenite in human lens epithelial cell lines. Overall, oral consumption of α-glucosyl hesperidin could delay the onset of selenite-induced cataract, at least in part by modulating the selenite-induced cell death in lens epithelial cells.

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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 （Pharmacological Reports）  71 （ 6 ） 1067 - 1078 2019年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  17341140

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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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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 2019年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00062952

　概要を見る

© 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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Oncogenic Ras mutant causes the hyperactivation of NF-κB via acceleration of its transcriptional activation

Tago K., Funakoshi-Tago M., Ohta S., Kawata H., Saitoh H., Horie H., Aoki-Ohmura C., Yamauchi J., Tanaka A., Matsugi J., Yanagisawa K.

Molecular Oncology （Molecular Oncology）  13 （ 11 ） 2493 - 2510 2019年11月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  15747891

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© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. It is well established that nuclear factor κB (NF-κB) acts as one of the most important transcription factors for tumor initiation and progression, as it both protects cells from apoptotic/necrotic signals and accelerates angiogenesis and tumor metastasis, which is mediated via the expression of target genes. However, it has not yet been clarified how oncogenic signals accelerate the activation of NF-κB. In the current study, we utilized untransformed NIH-3T3 cells stably harboring a κB-driven luciferase gene to show that an oncogenic mutant of Ras GTPase augmented TNFα-induced NF-κB activation. Notably, enforced expression of cyclin-dependent kinase inhibitors, such as p27Kip1 and p21Cip1, effectively canceled the accelerated activation of NF-κB, suggesting that oncogenic Ras-induced cell cycle progression is essential for the hyperactivation of NF-κB. Furthermore, we found that Ras (G12V) augmented the transcriptional activation of NF-κB, and this activation required the p38 MAP kinase. We observed that a downstream kinase of p38 MAP kinase, MSK1, was activated by Ras (G12V) and catalyzed the phosphorylation of p65/RelA at Ser-276, which is critical for its transcriptional activation. Significantly, phosphorylation of the p65/RelA subunit at Ser-276 was elevated in patient samples of colorectal cancer harboring oncogenic mutations of the K-Ras gene, and the expression levels of NF-κB target genes were drastically enhanced in several cancer tissues. These observations strongly suggest that oncogenic signal-induced acceleration of NF-κB activation is caused by activation of the p38 MAP kinase–MSK1 signaling axis and by cell cycle progression in cancer cells.

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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 2019年11月

研究論文（学術雑誌）, 共著, 査読有り,  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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The mechanisms of taxodione-induced apoptosis in BCR-ABL-positive leukemia cells

Uchihara Y., Tago K., Funakoshi-Tago M.

Folia Pharmacologica Japonica （Folia Pharmacologica Japonica）  153 （ 4 ） 147 - 154 2019年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00155691

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© 2019, Japanese Pharmacological Society. All rights reserved. Chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) are caused by a fusion protein, BCR-ABL, which induces cellular transformation by activating the signaling molecules, STAT5 and Akt. The specific BCR-ABL inhibitors including imatinib, nilotinib, and dasatinib, are clinically utilized in the treatment with CML and ALL patients. Although these BCR-ABL inhibitors are initially successful in the treatment of leukemia, many patients develop drug resistance due to the appearance of the gatekeeper mutation of BCR-ABL, T315I. Recently, we found that taxodione, a quinone methide diterpene isolated from a conifer, Taxodium distichum, significantly induced apoptosis in human myelogenous leukemia-derived K562 cells, which is positive for the bcr-abl gene. Taxodione reduced the activities of mitochondrial respiratory chain complex III, leading to the production of reactive oxygen species (ROS). An antioxidant agent, N-acetylcysteine (NAC), canceled taxodione-induced ROS production and apoptotic cell death, suggesting that taxodione induced apoptosis through ROS accumulation. Furthermore, in K562 cells treated with taxodione, BCR-ABL, STAT5 and Akt were sequestered in mitochondrial fraction, and their localization changes decrease their abilities to stimulate cell proliferation. Strikingly, NAC canceled these taxodione-caused inhibition of BCR-ABL, STAT5 and Akt. In addition, taxodione significantly induced apoptosis in transformed Ba/F3 cells by not only BCR-ABL but also T315I-mutated BCR-ABL through the generation of ROS, suggesting that taxodione has potential as anti-tumor drug with high efficacy to overcome BCR-ABL T315I mutation-mediated resistance in leukemia cells. It’s also expected that these knowledge becomes an important clue in the development of anti-cancer drugs against the broad range of tumors.

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

多胡 めぐみ

Experimental and Therapeutic Medicine （Experimental and Therapeutic Medicine）  17 （ 2 ） 1420 - 1425 2018年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  17920981

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© 2018, Spandidos Publications. All rights reserved. The lens has high concentrations of glutathione (GSH) and ascorbic acid (AsA) to maintain redox activity and prevent cataract formation, which is the leading cause of visual impairment worldwide. Metabolic syndrome is reported to be linked with a higher risk of age-associated cataract. As it was demonstrated previously that coffee consumption improved high-fat diet (HFD) -induced metabolic symptoms, it was hypothesized that coffee intake could delay the onset of obesity related-cataract; however, the effect of coffee consumption on this type of cataract remains unknown. Four-week-old male C57BL/6JJms SLC mice were divided into two groups and were provided ad libitum access to either a control diet (control groups) or a HFD (HFD groups). The control groups and HFD groups were further divided into three or four subgroups for each experiment. Coffee intake markedly reduced the increase in body weight in a roasting-time and concentration-dependent manner. Coffee consumption also prevented the HFD-induced decrease in the concentration of GSH and AsA, and treatment with pyrocatechol or caffeine also restored the reduction of antioxidant compounds. Plasma cholesterol and triglycerides were significantly higher in HFD groups; however, coffee brew or coffee constituent treatment in the HFD-fed mice group prevented elevation of these levels. Caffeine is a major coffee component and pyrocatechol is generated thought the roasting process. These results revealed that caffeine and pyrocatechol in coffee brew may be the key constituents responsible for preventing the reduction of lens GSH and AsA in HFD-fed animals.

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

多胡 めぐみ

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

多胡 めぐみ

Molecular Nutrition and Food Research （Molecular Nutrition and Food Research）  62 （ 21 ） e1800238 2018年09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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Hydroxytyrosol butyrate inhibits 6-OHDA-induced apoptosis through activation of the Nrf2/HO-1 axis in SH-SY5Y cells

多胡 めぐみ

European Journal of Pharmacology （European Journal of Pharmacology）  834   246 - 256 2018年07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. 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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A 5-hydroxyoxindole derivative attenuates LPS-induced inflammatory responses by activating the p38-Nrf2 signaling axis

多胡 めぐみ

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

多胡 めぐみ

Molecular Medicine Reports （Molecular Medicine Reports）  18 （ 1 ） 1043 - 1050 2018年05月

研究論文（学術雑誌）, 共著, 査読有り,  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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Taxodione induces apoptosis in BCR-ABL-positive cells through ROS generation

多胡 めぐみ

Biochemical Pharmacology （Biochemical Pharmacology）  154   357 - 372 2018年05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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