Faculty Profiles - NAKAMURA Yoshimasa

Papers - NAKAMURA Yoshimasa

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Papaya seed represents a rich source of biologically active isothiocyanate Reviewed

Yoshimasa Nakamura, Motoko Yoshimoto, Yoshiyuki Murata, Yasuaki Shimoishi, Yumi Asai, Eun Young Park, Kenji Sato, Yasushi Nakamura

JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 55 ( 11 ) 4407 - 4413 2007.5

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

In the present study, papaya (Carica papaya) seed and edible pulp were carefully separated and then the contents of benzyl isothiocyanate and the corresponding glucosinolate (benzyl glucosinolate, glucotropaeolin) quantified in each part. The papaya seed with myrosinase inactivation contained > 1 mmol of benzyl glucosinolate in 100 g of fresh seed. This content is equivalent to that of Karami daikon (the hottest Japanese white radish) or that of cress. The papaya seed extract also showed a very high activity of myrosinase and, without myrosinase inactivation, produced 460 mu mol of benzyl isothiocyanate in 100 g of seed. In contrast, papaya pulp contained an undetectable amount of benzyl glucosinolate and showed no significant myrosinase activity. The n-hexane extract of the papaya seed homogenate was highly effective in inhibiting superoxide generation and apoptosis induction in HL-60 cells, the activities of which are comparable to those of authentic benzyl isothiocyanate.

DOI： 10.1021/jf070159w

Selective cytotoxicity of benzyl isothiocyanate in the proliferating fibroblastoid cells Reviewed

Noriyuki Miyoshi, Koji Uchida, Toshihiko Osawa, Yoshimasa Nakamura

INTERNATIONAL JOURNAL OF CANCER 120 ( 3 ) 484 - 492 2007.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

In the present study, experiments using presynchronization culture cells demonstrated that benzyl ITC (BITC), previously isolated from a tropical papaya fruit extract, induced the cytotoxic effect preferentially in the proliferating human colon CCD-18Co cells to the quiescent ones. Quiescent CCD-18Co cells were virtually unaffected by BITC and marginal cytotoxicity was observed at 15 mu M. We observed that BITC dramatically induced the p53 phosphorylation and stabilization only in the quiescent (G(0)/G(1) phase-arrested) cells, but not significantly in the proliferating human colon CCD-18Co cells when compared with quiescent ones. We also observed ataxia telangiectasia-mutated (ATM) phosphorylation in the quiescent cells. The BITC-induced p53 phosphorylation was counteracted by caffeine treatment, implying the involvement of an ATM/ataxia telangiectasia and Rad3-related kinase signaling pathway. Moreover, downregulation of p53 by a siRNA resulted in the enhancement of susceptibility to undergo apoptosis by BITC. We also showed here that depletion of p53 abrogated G(0)/G(1), arrest accompanied by the declined expression of p21(waf1/cip1) and p27(kip1) in CCD-18Co cells. In conclusion, we identified p53 as a potential negative regulator of the apoptosis induction by BITC in the normal colon CCD-18Co cells through the inhibition of cell-cycle progression at the G(0)/G(1) phase. (c) 2006 Wiley-Liss, Inc.

DOI： 10.1002/ijc.22350

Benzyl isothiocyanate inhibits excessive superoxide generation in inflammatory leukocytes: implication for prevention against inflammation-related carcinogenesis Reviewed

N Miyoshi, S Takabayashi, T Osawa, Y Nakamura

CARCINOGENESIS 25 ( 4 ) 567 - 575 2004.4

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Inhibitors of excessive superoxide (O-2(-)) generation have been indicated to be more effective antioxidants than radical scavengers because O-2(-) anion is one of the precursors of several types of reactive oxygen species (ROS). We demonstrated here that benzyl isothiocyanate (BITC) is a potent inhibitor of leukocytic NADPH oxidase generating a great amount of O-2(-) in oxidative burst. The exposure of BITC to the differentiated HL-60 cells resulted in the inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced O-2(-) generation, while the methylthiocarbamate analog of BITC, hardly reactive with thiols including glutathione and protein sulfhydryl, did not show any effect. Pre-treatment of the cells with diethyl maleate significantly potentiated the BITC-induced inhibition, while pre-treatment with N-acetyl-cysteine counteracted it. These results led us to assume that a plausible intracellular target molecule(s) having a reactive sulfhydryl moiety might be regulated by the covalent attachment with BITC. In spite of no ability to affect the translocation of protein kinase C beta to the membrane, BITC probably modifies the electron transport system of cytochrome b(558), consistent with the observation that BITC inhibited the substrate utilization. Pre-treatments of mouse skin with BITC significantly attenuated the TPA-enhanced hydrogen peroxide level, suggesting that BITC indeed acts as an inhibitor of O-2(-) generation in mouse skin. A histological study also demonstrated that BITC inhibited TPA-induced leukocyte infiltration in the dermis. Because we have found several O-2(-) generation inhibitors to be effective chemopreventors against mouse skin carcinogenesis, the modifying effect of the topical application of BITC on TPA-induced mouse skin tumor promotion was investigated. We demonstrated for the first time that the pre-treatment with BITC 40 min prior to each TPA treatment significantly inhibited the number of papillomas per mouse. In conclusion, the results from this study provided biological evidence that BITC has a potential to prevent inflammation-related carcinogenesis including skin cancer.

DOI： 10.1093/carcin/bgh051

A link between benzyl isothiocyanate-induced cell cycle arrest and apoptosis: Involvement of mitogen-activated protein kinases in the Bcl-2 phosphorylation Reviewed

N Miyoshi, K Uchida, T Osawa, Y Nakamura

CANCER RESEARCH 64 ( 6 ) 2134 - 2142 2004.3

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC CANCER RESEARCH

In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis and the involvement of mitogen-activated protein kinases (MAPKs). The exposure of Jurkat human T-cell leukemia cells to BITC resulted in the inhibition of the G(2)-M progression that coincided with the apoptosis induction. The experiment using the phase-specific synchronized cells demonstrated that the G(2)-M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We also confirmed that BITC activated c-Jun N-terminal kinase (JNK) and p38 MAPK, but not extracellular signal-regulated kinase, at the concentration required for apoptosis induction. An experiment using a JNK-specific inhibitor SP600125 or a p38 MAPK inhibitor SB202190 indicated that BITC-induced apoptosis might be regulated by the activation of these two kinases. Conversely, BITC is likely to confine the Jurkat cells in the G(2)-M phase mainly through the p38 MAPK pathway because only the p38 MAPK inhibitor significantly attenuated the accumulation of inactive phosphorylated Cdc2 protein and the G(2)-M- arrested cell numbers. We reported here for the first time that the antiapoptotic Bcl-2 protein was phosphorylated by the BITC treatment without significant alteration of the Bcl-2 total protein amount. This was abrogated by a JNK specific inhibitor SP600125 at the concentration required for specific inhibition of the c-Jun phosphorylation. Moreover, the spontaneous phosphorylation of antiapoptotic Bcl-2 in the G(2)-M synchronized cells was enhanced synergistically by the BITC treatment. Involvement of the MAPK activation in the Bcl-2 phosphorylation and apoptosis induction also was observed in HL-60 and HeLa cells. Thus, we identified the phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with the JNK activation by BITC.

DOI： 10.1158/0008-5472.CAN-03-2296

Arachidonic acid cascade inhibitors modulate phorbol ester-induced oxidative stress in female ICR mouse skin: Differential roles of 5-lipoxygenase and cyclooxygenase-2 in leukocyte infiltration and activation Reviewed

Y Nakamura, M Kozuka, K Naniwa, S Takabayashi, K Torikai, R Hayashi, T Sato, H Ohigashi, T Osawa

FREE RADICAL BIOLOGY AND MEDICINE 35 ( 9 ) 997 - 1007 2003.11

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

We recently developed a short-term assay for skin H2O2 generation induced by double 12-O-tetradecanoylphorbol-13-acetate (TPA) applications for mechanistic study on skin epidermal carcinogenesis. In the present study, we investigated the individual roles of arachidonic acid metabolism in H2O2 generation in mouse skin inflammation. The experiments using inhibitors of arachidonic acid (AA) metabolism showed that corticosteroid and a lipoxygenase (LO) inhibitor expectedly suppressed double TPA application-induced H2O2 generation through the interference of chemotactic action but not by direct decomposition or scavenging. We also demonstrated that the treatment of AA (1 mumol) and 5-LO metabolites including leukotriene B-4 (LTB4) partly mimicked, though soybean LO-derived lipid hydroperoxide and prostaglandins did not, the priming effect evaluated by edema formation and leukocyte infiltration. We also confirmed that inflammatory leukocytes accumulated by LTB4 generated a significant amount of H2O2 by TPA stimulation. These results suggested that 5-LO metabolites of AA are the potential key molecules in the TPA-induced priming event. Interestingly, the cyclooxygenase (COX-) 2-selective inhibitor nimesulide (NS) and celecoxib (CXB) showed different responses than those of other inhibitors. These agents showed no specific potential to inhibit the priming event but significantly suppressed H2O2 generation, lipid peroxidation, and hyperplasia in mouse skin. From the results based on an in vitro leukocyte differentiation model, we speculated that the antioxidant effect of the COX-2 inhibitors might be partly associated with both counteraction of proinflammatory cytokine-enhanced ROS generation and inhibition of CD11b, an important molecule for cell adhesion, expression. Indeed, the topical application of NS attenuated the number of infiltrated leukocytes induced by TPA in mouse skin. Thus, these gathered data indicated the differential roles of 5-LO and COX-2 in leukocyte adhesion, infiltration, and H2O2 generation. (C) 2003 Elsevier Inc.

DOI： 10.1016/S0891-5849(03)00440-4

Pivotal role of electrophilicity in glutathione S-transferase induction by tert-butylhydroquinone Reviewed

Y Nakamura, T Kumagai, C Yoshida, Y Naito, M Miyamoto, H Ohigashi, T Osawa, K Uchida

BIOCHEMISTRY 42 ( 14 ) 4300 - 4309 2003.4

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

Although the induction of glutathione S-transferase (GST) activity by tert-butylhydroquinone (tBHQ) has been well-documented in several cell culture systems and rodent experiments, the exact mechanism responsible for its inducibility is still not thoroughly understood. To more precisely define the molecular mechanism of GST induction by tBHQ, we examined the one-electron oxidation and glutathione (GSH) reaction potentials of tBHQ as compared to its analogue, 2,5-di-tert-butylhydroquinone (DtBHQ). tBHQ and DtBHQ showed similar one-electron oxidation potentials, including free radical quenching (antioxidant), oxidative conversion of both compounds to a benzoquinone form, and Cu2+-dependent superoxide generation. On the other hand, the reduced GSH level was observed by the addition of tBHQ, but not DtBHQ, suggesting that tBHQ acts as an electrophile while DtBHQ does not. The data were consistent with the observation that tBHQ more potently induced the GSTP1 gene expression in RL34 cells than DtBHQ did. Moreover, we indeed detected the GSH-tBHQ conjugates in the cells exposed to tBHQ using an electrochemical detector-high-performance liquid chromatography technique. Thus, we conclude that an electrophilic quinone oxidation product that reacts with intracellular nucleophiles including protein thiol or GSH plays a major role in the GSTP1 gene expression.

DOI： 10.1021/bi0340090

Involvement of the mitochondrial death pathway in chemopreventive benzyl isothiocyanate-induced apoptosis Reviewed

Y Nakamura, M Kawakami, A Yoshihiro, N Miyoshi, H Ohigashi, K Kawai, T Osawa, K Uchida

JOURNAL OF BIOLOGICAL CHEMISTRY 277 ( 10 ) 8492 - 8499 2002.3

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

In the present study, we studied the molecular mechanism underlying cell death induced by a cancer chemoprotective compound benzyl isothiocyanate (BITC). The cytotoxic effect of BITC was examined in rat liver epithelial RL34 cells. Apoptosis was induced when the cells were treated with 20 muM BITC, characterized by the appearance of phosphatidylserine on the outer surface of the plasma membrane and caspase-3 activation, whereas no caspase activation and propidium iodide incorporation into cell were detected with 50 muM BITC that induced necrosis. The mitochondrial death pathway was suggested to be involved in BITC-induced apoptosis because the treatment of cells with BITC-induced caspase-9-dependent apoptosis and mitochondrial transmembrane potential (DeltaPsim) alteration. We demonstrated here for the first time that BITC directly modifies mitochondrial functions, including inhibition of respiration, mitochondrial swelling, and release of cytochrome c. Moreover, glutathione depletion by diethyl maleate significantly accelerated BITC-triggered apoptosis, suggesting the involvement of a redox-dependent mechanism. This was also implicated by the observations that intracellular accumulation of reactive oxygen species, including superoxide (O-2(.-)) and hydroperoxides (HPOs), was indeed detected in the cells treated with BITC and that the intracellular HPO level was significantly attenuated by pretreatment with N-ace-tylcysteine. The treatment with a pharmacological scavenger of 07 Tiron, also diminished the HPO formation by similar to80%,c, suggesting that most of the HPOs were H2O2 derived from the dismutation of O-2(.-). These results suggest that BITC induces apoptosis through a mitochondial redox-sensitive mechanism.

DOI： 10.1074/jbc.M109760200

Ebselen, a glutathione peroxidase mimetic seleno-organic compound, as a multifunctional antioxidant - Implication for inflammation-associated carcinogenesis Reviewed

Y Nakamura, Q Feng, T Kumagai, K Torikai, H Ohigashi, T Osawa, N Noguchi, E Niki, K Uchida

JOURNAL OF BIOLOGICAL CHEMISTRY 277 ( 4 ) 2687 - 2694 2002.1

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Ebselen, a seleno-organic compound showing glutathione peroxidase-like activity, is one of the promising synthetic antioxidants. In the present study, we investigated the antioxidant activities of ebselen using a 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated mouse skin model. Double pretreatments of mouse skin with ebselen significantly inhibited TPA-induced formation of thiobarbituric acid-reacting substance, known as an overall oxidative damage biomarker, in mouse epidermis, suggesting that ebselen indeed acts as an antioxidant in mouse skin. The andoxidative effect of ebselen is attributed to its selective blockade of leukocyte infiltration and activation leading to attenuation of the H2O2 level. In in vitro studies, ebselen inhibited TPA-induced superoxide generation in differentiated HL-60 cells and lipopolysaccharide-induced cyclooxygenase-2 protein expression in RAW 264.7 cells. In addition, we demonstrated for the first time that ebselen potentiated phase II enzyme activities, including NAD(P)H:(quinone-acceptor) oxidoreductasel and glutathione S-transferase in cultured hepatocytes and in mouse skin. These results strongly suggest that ebselen, a multifunctional antioxidant, is a potential chemopreventive agent in inflammation-associated carcinogenesis.

DOI： 10.1074/jbc.M109641200

A catechol antioxidant protocatechuic acid potentiates inflammatory leukocyte-derived oxidative stress in mouse skin via a tyrosinase bioactivation pathway Reviewed

Y Nakamura, K Torikai, H Ohigashi

FREE RADICAL BIOLOGY AND MEDICINE 30 ( 9 ) 967 - 978 2001.5

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

The modifying effects of topical application of a catechol antioxidant protocatechuic acid (PA) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory responses in mouse skin were investigated. Treatment with a high dose (20,000 nmol) of PA, based on time of application, modifies inflammatory responses in the skin of the B6C3F(1) mouse, a resistant strain to inflammatory response induction by TPA, but shows much higher tyrosinase expression than that of an albino mouse. The responsibility of a large amount of PA-induced leukocyte infiltration to an inflamed region in a B6C3F(1) mouse is mon sensitive than that of an ICR albino mouse. When ICR mice were treated with TPA (1.6 nmol) twice weekly for 5 weeks to induce chronic inflammatory responses, pretreatment with 1600 nmol PA 30 min prior to each TPA treatment significantly enhanced the inflammatory responses including edema formation, leukocyte infiltration, and the level of thiobarbituric acid-reacting substances. The dose-dependency was closely parallel to the results of a tumor promotion study of PA previously reported. Further, the treatment of PA alone resulted in tyrosinase-dependent contact hypersensitivity in ICR mouse skin. In addition, the in vitro study of cytotoxicity demonstrated that bioactivation by tyrosinase but not myeloperoxidase of PA significantly enhanced cytotoxicity and intracellular glutathione consumption. We conclude that the tyrosinase-derived reactive quinone intermediate(s) of PA, which binds nucleophilic residues of proteins including sulfhydryl group and conjugates of which are recognized as haptens, was partially involved in alteration of the cellular immune functions including oxygen radical-generating leukocytes migration to inflamed regions. (C) 2001 Elsevier Science Inc.

DOI： 10.1016/S0891-5849(01)00481-6

A simple phenolic antioxidant protocatechuic acid enhances tumor promotion and oxidative stress in female ICR mouse skin: dose- and timing-dependent enhancement and involvement of bioactivation by tyrosinase Reviewed

Y Nakamura, K Torikai, Y Ohto, A Murakami, T Tanaka, H Ohigashi

CARCINOGENESIS 21 ( 10 ) 1899 - 1907 2000.10

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

The modifying effects of topical application of the phenolic antioxidant protocatechuic acid (PA) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin tumor promotion were investigated. Dimethylbenz[a]anthracene-initiated female ICR mice were treated with TPA (1.6 nmol) twice weekly for 20 weeks to promote papilloma formation. Pre-treatment with 16 nmol PA 30 min prior to each TPA treatment significantly inhibited the number of papillomas per mouse by 52% (P < 0.05). On the other hand, PA pre-treatment at a high dose (1600 nmol) significantly enhanced tumor numbers by 38% (P < 0.05). Interestingly, in the group treated with a quite high dose (20000 nmol) of PA 5 min prior to each TPA application, the average number of tumors per mouse was reduced by 38%, whereas the same PA dose 3 h before TPA treatment significantly enhanced tumor numbers by 84% (P < 0.01). These results suggested that topically applied PA was converted to compound(s) lacking antioxidative properties and/or rather possessing the potential to enhance tumor development. A similar tendency was also observed in the short-term experiment of TPA-induced inflammation and oxidative stress; i.e. two groups pretreated with PA at 20 000 nmol, 30 min and 3 h before TPA treatment, did not show suppression or even significantly enhanced TPA-induced leukocyte infiltration, H2O2 generation, thiobarbituric acid-reacting substances level and proliferating cell nuclear antigen index, while PA treatment together with TPA significantly suppressed these parameters. Treatment with a high dose (20 000 nmol) of PA alone for 3 h enhanced oxidative stress by reducing glutathione levels in mouse skin, which was counteracted by the tyrosinase inhibitor arbutin, Oxidative stress responses such as leukocyte infiltration and H2O2 generation were also counteracted by arbutin, These results suggested that tyrosinase-dependent oxidative metabolism of PA was at least partially involved in the enhanced effects of PA on TPA-induced inflammatory responses and thus tumor promotion.

DOI： 10.1093/carcin/21.10.1899

A glutathione S-transferase inducer from papaya, rapid screening, identification and structure-activity relationship of isothiocyanates Reviewed

Y Nakamura, Y Morimitsu, T Uzu, H Ohigashi, A Murakami, Y Naito, Y Nakagawa, T Osawa, K Uchida

CANCER LETTERS 157 ( 2 ) 193 - 200 2000.9

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCI IRELAND LTD

We have developed a simple system for rapid detection and measurement of glutathione S-transferase placental form (GSTP1) that detoxify polycyclic aromatic hydrocarbons using the cultured rat normal liver epithelial cell line, (RL34) cells. Survey of fruit extracts for GST inducing ability identified both papaya and avocado as significant sources. Benzyl isothiocyanate (BITC) was isolated from papaya methanol extract as a principal inducer of GST activity. Further, the GST inducing ability of a total of 20 isothiocyanates (ITCs) and their derivatives was investigated. Some ITCs showed significant induction, and BITC was one of the most potent inducers among all compounds tested in the present study. The modification of isothiocyanate group (-NCS) or introduction of substituent group to the a-carbon modifies GST induction. Moreover, a significant correlation (P < 0.01, r = 0.913) between the GST activity enrichment and GSTP1 protein induction by ITCs was observed. We also indicated that phenethyl ITC and nitrophenyl ITC, potently inducing GST activity, but not inactive benzyl isocyanate, are potential inducers of intracellular reactive oxygen intermediates (ROIs). Our system of GSTP1 induction is appropriate for the chemical research such as screening and identification of novel type of inducers as well as the structure-activity relationship studies, providing mechanistic insight into essential structural elements for GSTP1 induction. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0304-3835(00)00487-0

Redox regulation of glutathione S-transferase induction by benzyl isothiocyanate: Correlation of enzyme induction with the formation of reactive oxygen intermediates Reviewed

Y Nakamura, H Ohigashi, S Masuda, A Murakami, Y Morimitsu, Y Kawamoto, T Osawa, M Imagawa, K Uchida

CANCER RESEARCH 60 ( 2 ) 219 - 225 2000.1

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC CANCER RESEARCH

Here we report the molecular mechanism underlying the induction of glutathione S-transferase (GST) in rat liver epithelial RL34 cells treated with a cancer chemopreventive isothiocyanate compound, benzylisothiocyanate (BITC), BITC was found to significantly induce GST activity in RL34 cells. Northern and Western blot analyses demonstrated that BITC specifically enhanced the production of the class pi GST isozyme (GSTP1), Our studies demonstrated for the first time that the addition of BITC to the cells resulted in an immediate increase in the reactive oxygen intermediates (ROIs) detected by a fluorescence probe, 2',7'-dichlorofluorescin diacetate. The level of the ROIs in the cells treated with BITC (10 mu M) was similar to 50-fold higher than those in the control cells. Furthermore, glutathione depletion by diethyl maleate significantly enhanced BITC-induced ROI production and accelerated the BITC-induced elevation of the GST activity, whereas pretreatment of the cells with glutathione inhibited both the ROI production and GST induction. The structure-activity relationship of the isothiocyanates also indicated that the ROI-producing activities closely correlated with their GST-inducing potencies. Moreover, the GSTP1 enhancer I-containing region was found to be essential for induction of the GSTP1 gene by intracellular ROI inducers such as BITC and diethyl maleate. These data suggest the involvement of the redox regulation on the induction of GSTP1 by BITC.

Suppression of tumor promoter-induced oxidative stress and inflammatory responses in mouse skin by a superoxide generation inhibitor 1 '-acetoxychavicol acetate Reviewed

Y Nakamura, A Murakami, Y Ohto, K Torikai, T Tanaka, H Ohigashi

CANCER RESEARCH 58 ( 21 ) 4832 - 4839 1998.11

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC CANCER RESEARCH

Double applications of phorbol eaters trigger excessive reactive oxygen species (ROS) production in mouse skin. previously reported data suggest that the two applications induce distinguishable biochemical events, namely, priming and activation. The former is characterized as a recruitment of inflammatory cells, such as neutrophils, by chemotactic factors to inflammatory regions and edema formation. The latter is responsible for ROS generation. Thus, inhibitory effects of 1'-acetoxychavicol acetate (ACA), previously reported to be a superoxide generation inhibitor is vitro, on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative stress and inflammatory responses in mouse skin model were examined using a double application of ACA. We demonstrated that two pretreatments and pretreatment with ACA (810 mmol) in the activation phase suppressed double TPA application-induced H2O2 formation in mouse skin, ACA exhibited no inhibitory effects on edema formation and the enhancement of myeloperoxidase activity during the first TPA treatment, whereas the anti-inflammatory agent genistein administered at the same dose inhibited both biomarkers, No inhibitory potential of ACA for TPA-induced H2O2 formation in the priming phase was confirmed. On the other hand, in the in vitro study, ACA inhibited ROS generation in differentiated HL-60 cells more strongly than did 1'-hydroxychavicol, which showed no inhibition by pretreatment in the activation phase In addition, allopurinol did not inhibit double TPA application-induced H2O2 formation in mouse skin, These findings suggest that the NADPH oxidase system of neutrophils rather than the epithelial xanthine oxidase system is dominant for the O-2(-)-generating potential in double TPA-treated mouse skin. ACA significantly inhibited mouse epidermis thiobarbituric acid-reacting substance formation, known as an overall oxidative damage biomarker, Moreover, histological studies demonstrated that ACA inhibited double TPA treatment-induced morphological changes reflecting inflammatory response, such as edema formation, leukocyte infiltration, hyperplasia, and cell proliferation. Furthermore, pretreatment with ACA but not 1'-hydroxychavicol in the activation phase inhibits double TPA application-induced increases in both number of leukocytes and proliferating cell nuclear antigen index. These results suggested that ROS from leukocytes including O-2(-) plays an important role for continuous and excessive production of chemotactic factors, leading to chronic inflammation and hyperplasia, which are inhibitable by ACA. Thus, we concluded that O-2(-) generation inhibitors are agents that effectively inhibit oxidative stress and inflammatory responses in mouse skin.

Inhibitory effects of curcumin and tetrahydrocurcuminoids on the tumor promoter-induced reactive oxygen species generation in leukocytes in vitro and in vivo Reviewed

Yoshimasa Nakamura, Yoshimi Ohto, Akira Murakami, Toshihiko Osawa, Hajime Ohigashi

Japanese Journal of Cancer Research 89 ( 4 ) 361 - 370 1998

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：Japanese Cancer Association

The inhibitory effects of curcumin and two tetrahydrocurcuminoids on tumor promoter-induced oxidative stress in vitro and in vivo were investigated. Curcumin, tetrahydrocurcumin (THC) and dihydroxytetrahydrocurcumin (DHTHC) exhibited significant inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced O2 - generation in differentiated HL-60 cells. The inhibitory activity of THC was weaker than that of curcumin. This tendency was the inverse of the results of previous studies on in vitro antioxidative activity against lipid peroxidation. The curcuminoids inhibited TPA-induced intracellular peroxide formation in differentiated HL-60 cells. THC exhibited much weaker inhibition of intracellular peroxide formation than curcumin, suggesting that this inhibition might be attributable to the inhibition of O2 - generation. The inhibitory effects of curcuminoids on TPA-induced H2O2 formation in female ICR mouse skin were further examined using the double-TPA-application model. Each TPA application induces two distinct biochemical events, 1) recruitment of inflammatory cells to the inflammatory regions and 2) activation of oxidant-producing cell. Double pretreatment of mice with curcuminoids before each TPA treatment significantly suppressed double TPA application-induced H2O2 formation in the mouse skin. Coadministrations of curcumin with either first or second TPA treatment significantly inhibited H2O2 formation. In addition, THC tends to show weaker inhibitory activities than curcumin in bioassays related to tumor promotion, i.e., inhibition of tumor promoter-induced inflammation in mouse skin and Epstein-Barr virus activation. These tendencies were parallel to those in the tumor-suppressive potential of curcumin and THC in mouse skin, as previously reported. Thus, we concluded that curcuminoids significantly suppress TPA-induced oxidative stress via both interference with infiltration of leukocytes into the inflammatory regions and inhibition of their activation.

DOI： 10.1111/j.1349-7006.1998.tb00572.x

Inhibitory effect of pheophorbide a, a chlorophyll-related compound, on skin tumor promotion in ICR mouse Reviewed

Y Nakamura, A Murakami, K Koshimizu, H Ohigashi

CANCER LETTERS 108 ( 2 ) 247 - 255 1996.11

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCI IRELAND LTD

Anti-tumor-promoting activity of pheophorbide a (PPBa), a chlorophyll-related compound, was examined in a two-stage carcinogenesis experiment in ICR mouse skin by 7,12-dimethylbenz[a]anthracene (DMBA, 0.19 mu mol) and 12-O-tetradecanoylphorbol-13-acetate (TPA, 1.6 nmol). Topical application of PPBa (160 nmol) markedly reduced the average number of tumors per mouse and the ratio of tumor-bearing mice (inhibitory ratio: IR = 56%, P < 0.01 and 31%, P < 0.005, respectively). PPBa exhibited potent anti-inflammatory activity in ICR mouse ears and moderate inhibitory activity toward TPA-induced superoxide (O-2(-)) generation in differentiated HL-60 cells. While CuPPBa, a synthetic copper complex of PPBa, exhibited higher anti-inflammatory activity than that of indomethacin, it showed little antioxidative effect against formation of lipid hydroperoxides (LOOHs) and malondialdehyde (MDA), suggesting that the antioxidative effect of PPBa might not be important for anti-inflammatory activity. These results imply that the active mechanism of PPBa for anti-tumor promotion might be partly involved in inhibition of TPA-induced inflammatory responses by suppressing leukocyte activation.

DOI： 10.1016/S0304-3835(96)04422-9

Involvement of reactive carbonyl species in inhibition of germination and seedling growth by salt stress in rice Invited

Faruk Hossain Khan, Yoshitaka Nakashima, Toshiyuki Nakamura, Yoshimasa Nakamura, Yoshihiko Hirai, Md Anamul Hoque, Mohammad Saidur Rhaman, Shintaro Munemasa, Jun'ichi Mano, Yoshiyuki Murata

Bioscience, Biotechnology, and Biochemistry 2025.5

Proteome analysis reveals the molecular mechanisms of fucoxanthin-induced apoptosis in glial cells PC12 after nanoencapsulation Reviewed

Chunyan Wang, Xinxin Ma, Yoshimasa Nakamura, Hang Qi

Journal of Functional Foods 126 2025.3

Publishing type：Research paper (scientific journal)

Fucoxanthin (FX) is a natural pigment belonging to the xanthophyll carotenoid, which has been proven to have inhibitory effects on glioblastoma. In our previous study, nano-FX was fabricated through whey protein and flaxseed gum co-encapsulated for improved stability and glial cell (PC12) inhibitory activity of FX. To investigate the molecular mechanisms of FX-induced apoptosis in PC12 after nanoencapsulation, proteomics analysis based on TMT technology was performed. Protein profiles in PC12 were determined after 0 (Control), 8 (L_Treat), and 24 (H_Treat) μg/mL nano-FX intervention. Results indicated 78 differential abundant proteins (DAPs) linked to the cytotoxicity of nano-FX could be potential biomarkers. GO and KEGG analysis revealed altered proteasome and NF-kappa B/MAPK/TNF. Proteins involved in cell proliferation were downregulated, while proteins response to autophagy and apoptosis were upregulated. The multiple mechanisms involved in the anti-tumor activity of nano-FX will provide a reference for the development of FX high value-added functional foods.

DOI： 10.1016/j.jff.2025.106710

Fraglide-1 from traditional Chinese aromatic vinegar: A natural AhR antagonist for atopic dermatitis. Reviewed International journal

Kosuke Kato, Miki Akamatsu, Saya Kakimaru, Mayuko Koreishi, Masahiro Takagi, Masahiro Miyashita, Yoshiyuki Murata, Yoshimasa Nakamura, Ayano Satoh, Yoshio Tsujino

Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 115301 - 115301 2025.2

Language：English Publishing type：Research paper (scientific journal)

Traditional Chinese Zhenjiang aromatic vinegar (Kozu) contains Fraglide-1 (FG1), a bioactive lactone with demonstrated peroxisome proliferator-activated receptor gamma (PPARγ) agonist and antioxidant activities. This study explored FG1's novel ability to antagonize the aryl hydrocarbon receptor (AhR) signaling pathway, which regulates artemin expression and contributes to itching and inflammation in atopic dermatitis. Through molecular docking simulations and cell-based assays in human keratinocytes, we demonstrated FG1's potent antagonistic activity against AhR signaling. FG1 effectively suppressed FICZ-induced inflammatory responses, including artemin expression, with potency (half maximal inhibitory concentration, IC50 = 5.1 μM) comparable to the synthetic antagonist StemRegenin 1 (SR1) while demonstrating a superior safety profile (median lethal concentration, LC50 > 100 μM vs. 27.5 μM for SR1). These findings expand our understanding of bioactive compounds from traditional fermented foods and their regulatory effects on AhR signaling, providing a foundation for future studies on FG1's role in modulating skin inflammation.

DOI： 10.1016/j.fct.2025.115301

Dual role of cytosolic GSH in the ABA signaling pathway and plasma membrane ion channel regulation in guard cells of Vicia faba. Reviewed International journal

Huifei Yin, Toshiyuki Nakamura, Yoshimasa Nakamura, Shintaro Munemasa, Yoshiyuki Murata

Journal of plant physiology 306 154447 - 154447 2025.2

Language：English Publishing type：Research paper (scientific journal)

Abscisic acid (ABA) induces stomatal closure in higher plants under drought stress. Glutathione (GSH) negatively regulates ABA-induced stomatal closure and reactive carbonyl species (RCS) play a role as signal mediators downstream of reactive oxygen species production in ABA signaling pathway in Arabidopsis thaliana. Activation of slow (S-type) anion channels and inhibition of inward-rectifying potassium ion (Kin+) channels in the plasma membrane are essential for ABA-induced stomatal closure. However, there is limited evidence regarding role of GSH in the activation of S-type anion channels and the inhibition of Kin+ channels. We used Vicia faba to clarify the regulation of these ion channels by GSH and RCS. Pretreatment of guard-cell protoplasts with the GSH-supplementing agent, glutathione monoethyl ester (GSHmee), suppressed the activation of S-type anion channels and the inactivation of Kin+ channels induced by ABA. The pretreatment with the RCS scavenger carnosine suppressed the activation of S-type anion channels and the inactivation of Kin+ channels by ABA. On patch clamping guard-cell protoplasts, the addition of GSH to the pipette (cytosolic) buffer decreased the S-type anion currents and increased the Kin+ currents. These results suggest that cytosolic GSH is involved in ABA-induced stomatal closure via negative regulation of ABA signaling and via direct regulation of ion channel activities in V. faba.

DOI： 10.1016/j.jplph.2025.154447

TGG1 and TGG2 mutations impair allyl isothiocyanate-mediated stomatal closure in Arabidopsis thaliana. Reviewed International journal

Kadri Oumaima, Mohammad Shakhawat Hossain, Wenxiu Ye, Eiji Okuma, Mohammad Issak, Mohammad Mahbub Islam, Misugi Uraji, Yoshimasa Nakamura, Izumi C Mori, Shintaro Munemasa, Yoshiyuki Murata

Protoplasma 2025.2

Language：English Publishing type：Research paper (scientific journal)

Myrosinase, referred to as thioglucoside glucohydrolase (TGG), plays a crucial role in plant physiology through catalyzing the hydrolysis of glucosinolates into bioactive isothiocyanates. In Arabidopsis thaliana, the myrosinases TGG1 and TGG2 are essential for abscisic acid- and methyl jasmonate-induced stomata closure. Allyl isothiocyanate (AITC), one of myrosinase products, triggers stomatal closure in A. thaliana. We investigated stomatal responses to AITC to clarify the role of TGG1 and TGG2 in Arabidopsis guard-cell signaling. Allyl isothiocyanate at 50 μM and 100 μM induced stomatal closure in the tgg1 and tgg2 single mutants but not in the tgg1 tgg2 double mutant. Furthermore, AITC at 50 μM induced the production of reactive oxygen species and nitric oxide, cytosolic alkalization, and oscillations in cytosolic free calcium concentration in guard cells of both wild-type and mutant plants. These findings suggest that TGG1 and TGG2 are involved in AITC signaling pathway through interaction with signal component(s) downstream of these signaling events, which is not accompanied by hydrolysis of glucosinolates because of the difference in subcellular localization between enzymes (myrosinases) and substrates (glucosinolates).

DOI： 10.1007/s00709-025-02039-z

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