Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Hericenone C is one of the most abundant secondary metabolites derived from Hericium erinaceus, under investigation for medicinal properties. Here, we report that Hericenone C inhibits the second phase of formalin-induced nociceptive behavior in mice. As the second phase is involved in inflammation, in a mechanistic analysis on cultured cells targeting NF-κB response element (NRE): luciferase (Luc)-expressing cells, lipopolysaccharide (LPS)-induced NRE::Luc luciferase activity was found to be significantly inhibited by Hericenone C. Phosphorylation of p65, which is involved in the inflammatory responses of the NF-κB signaling pathway, was also induced by LPS and significantly reduced by Hericenone C. Additionally, in mice, the number of CD11c-positive cells increased in the paw during the peak of the second phase of the formalin test, which decreased upon Hericenone C intake. Our findings confirm the possibility of Hericenone C as a novel therapeutic target for pain-associated inflammation.

DOI： 10.1016/j.bbrc.2024.150077

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

Background and purpose

Maintaining mitochondrial quality is attracting attention as a new strategy to treat diabetes and diabetic complications. We previously reported that mitochondrial hyperfission by forming a protein complex between dynamin‐related protein (Drp) 1 and filamin, mediates chronic heart failure and cilnidipine, initially developed as an L/N‐type Ca²⁺ channel blocker, improves heart failure by inhibiting Drp1‐filamin protein complex. We investigated whether cilnidipine improves hyperglycaemia of various diabetic mice models.

Experimental Approach

Retrospective analysis focusing on haemoglobin A1c (HbA1c) was performed in hypertensive and hyperglycaemic patients taking cilnidipine and amlodipine. After developing diabetic mice by streptozotocin (STZ) treatment, an osmotic pump including drug was implanted intraperitoneally, followed by weekly measurements of blood glucose levels. Mitochondrial morphology was analysed by electron microscopy. A Ca²⁺ channel‐insensitive cilnidipine derivative (1,4‐dihydropyridine [DHP]) was synthesized and its pharmacological effect was evaluated using obese (ob/ob) mice fed with high‐fat diet (HFD).

Key Results

In patients, cilnidipine was superior to amlodipine in HbA1c lowering effect. Cilnidipine treatment improved systemic hyperglycaemia and mitochondrial morphological abnormalities in STZ‐exposed mice, without lowering blood pressure. Cilnidipine failed to improve hyperglycaemia of ob/ob mice, with suppressing insulin secretion. 1,4‐DHP improved hyperglycaemia and mitochondria abnormality in ob/ob mice fed HFD. 1,4‐DHP and cilnidipine improved basal oxygen consumption rate of HepG2 cells cultured under 25 mM glucose.

Conclusion and implications

Inhibition of Drp1‐filamin protein complex formation becomes a new strategy for type 2 diabetes treatment.

DOI： 10.1111/bph.16487

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

The retina is the only organ projecting external light to the suprachiasmatic nucleus. Cholecystokinin receptor type A (Cckar/Cckar) is one of the essential factors for light reception in retinal cells. As there was a lack of literature on the matter, we aimed to elucidate the cause of the time-dependent phase change in clock gene expression. We found that Cckar mRNA expression in retinal cells exhibited diurnal variations. The rhythm of expression of the clock gene Per1/Per2 in retinal cells was altered in Cckar -/- mice. The light sensitivity of retinal cells was evaluated in wild-type mice, which showed c-Fos was activated in the ganglion cell layer more than in the inner granular layer. This increase in the number of c-Fos-positive cells was suppressed by lorglumide, a Cckar antagonist. Treatment of rat retina primary cells with lorglumide suppressed Per2 transcription, which was altered in a time-dependent manner relative to the Per2 expression. Light irradiation studies in Cckar -/- mice did not exhibit an increase in Period expression in the suprachiasmatic nucleus. These results indicate that Cckar is among the factors that regulate the cycle of clock genes on the retina. Cckar knockout attenuates the light responsiveness of suprachiasmatic nucleus and reduces the expression amplitude of Period genes in the retina. Thus, Cckar may contribute to entrainment of the light environment and maintenance of the expression cycle of Period gene, which is one of the core clock genes.

DOI： 10.1016/j.heliyon.2024.e32653

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Neuropathic pain often results from injuries and diseases that affect the somatosensory system. Disruption of the circadian clock has been implicated in the exacerbation of the neuropathic pain state. However, in this study, we report that mice deficient in a core clock component Period2 (Per2m/m mice) fail to develop tactile pain hypersensitivity even following peripheral nerve injury. Similar to male wild-type mice, partial sciatic nerve ligation (PSL)-Per2m/m male mice showed activation of glial cells in the dorsal horn of the spinal cord and increased expression of pain-related genes. Interestingly, α1D-adrenergic receptor (α1D-AR) expression was up-regulated in the spinal cord of Per2m/m mice, leading to increased production of 2-arachidonoylglycerol (2-AG), an endocannabinoid receptor ligand. This increase in 2-AG suppressed the PSL-induced tactile pain hypersensitivity. Furthermore, intraspinal dorsal horn injection of adeno-associated viral vectors expressing α1D-AR also attenuated pain hypersensitivity in PSL-wild-type male mice by increasing 2-AG production. Our findings reveal an uncovered role of the circadian clock in neuropathic pain disorders and suggest a link between α1D-AR signaling and the endocannabinoid system.

DOI： 10.1093/pnasnexus/pgad482

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

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代謝活性を検出することで、細胞固有の代謝状態を明らかにし、細胞の恒常性と増殖の根底にあるメカニズムを解明することができる。しかし、代謝経路の研究のための蛍光アプローチは、まだほとんど未開拓である。そこで我々は、細胞や組織における脂質異化の重要なプロセスである脂肪酸β酸化（FAO）を蛍光ベースで検出するための新しい化学プローブを開発した。このプローブはFAOの基質となり、代謝反応の結果、反応性のキノン・メチド（QM）を形成する。遊離したQMは細胞内タンパク質に共有結合で捕捉され、その後の蛍光団との生物学的直交ライゲーションにより蛍光分析が可能となる。この反応ベースのセンシングにより、蛍光イメージング、ゲル内蛍光活性ベースのタンパク質プロファイリング（ABPP）、蛍光活性化セルソーティング（FACS）などの多様な分析技術を用いて、細胞内のFAO活性を所望の発光波長で検出することが可能になった。このプローブは、培養細胞中の化学修飾物質によって誘導されるFAO活性の変化を検出することができた。さらに、このプローブをマウス肝組織におけるFAOの蛍光イメージングに用い、FACSと遺伝子発現解析を組み合わせることにより、肝細胞におけるFAO活性の代謝的不均一性を明らかにし、脂肪酸代謝研究のための化学的ツールとしての本プローブの有用性を強調した。

DOI： 10.1021/jacs.3c02043

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

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Efficient and practical construction of deuterated molecules has been a long-standing challenge. Although α-deutero carboxylic acids are ubiquitous structural components that serve as a wide array of synthons, and are thus in high demand, α-selective and mild methods for the deuteration of carboxylic acids have remained unexplored. Here we report the development of a ternary catalytic system for the α-deuteration of carboxylic acids in high yields with excellent levels of α-deuteration. The method shows wide functional group tolerance, including the late-stage deuteration of complex molecules, pharmaceuticals and natural products. Mechanistic studies and pK a calculations indicate that the reaction proceeds through the enolization of an acyl pyridinium species. The process was applied to the synthesis of a deuterated EP3 receptor antagonist, with the deutero analogue showing increased metabolic stability in human microsomes compared with the proto compound. [Figure not available: see fulltext.]

DOI： 10.1038/s44160-022-00139-9

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Other Link： https://www.nature.com/articles/s44160-022-00139-9

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

がん細胞における概日時計機構の破壊は、腫瘍の悪性化に関与している。がん治療に関する研究から、乳がん腫瘍には乳がん幹細胞様細胞（BCSC）を含む異種細胞が存在することが明らかになっている。BCSCsはしばしば高いアルデヒド脱水素酵素（ALDH）活性を特徴とし、がんの悪性化に関連している。本研究では、マウス乳がん4T1細胞において、主要な概日リズム成分であるCLOCKがALDH活性を負に制御していることを明らかにした。高ALDH活性の4T1細胞ではCLOCKの発現が抑制され、CLOCKの発現を増強すると、腫瘍形成能や浸潤能といった幹細胞としての性質が消失した。さらに、高ALDH活性4T1におけるCLOCKの発現低下は、マイクロRNA：miR-182によって転写後制御されていた。miR-182をノックアウトするとCLOCKの発現が回復し、腫瘍の増殖が抑制された。この知見は、転写後制御を標的としたBCSCsにおけるCLOCKの発現増加が、幹細胞性に関連した悪性腫瘍を克服することを示唆しており、乳がん治療の新たな戦略となる可能性がある。

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

九州大学大学院薬学研究院の「薬薬連携研究」の成果として、慢性腎臓病 (CKD)時に生じる心臓の炎症/線維化がビタミンAと単球内の概日時計変容を介して悪化する仕組みを明らかにしました。慢性腎臓病 (CKD)による腎機能の低下は様々な合併症を引き起こしますが、特に発症頻度が高いのが心疾患です。にもかかわらず、CKD時に心疾患リスクが上昇する要因については十分理解されておらず、末期CKD患者の死因1位は心不全です。一方、多くの生物は、地球の自転に伴う外部環境の周期的な変化に対応するため、自律的にリズムを発振する機能 (体内時計)を保持しています。この体内時計は時計遺伝子と呼ばれる複数の転写因子によって維持されていますが、疾患時には時計遺伝子の発現/機能にしばしば異常が認められます。今回、研究グループはマウスおよびヒト血液検体を用いた実験により、CKD時に生じる血中ビタミンA濃度の上昇が時計遺伝子の発現を変容させること、およびこの発現変容が単球の炎症促進作用を高めることを突き止めました。また、この単球が心臓に浸潤することで、心臓の炎症を悪化させることも明らかにしました。これらの知見から、CKD時に単球の炎症活性を上昇させる分子を標的とした治療薬の開発や、ビタミンAの異常な蓄積を改善する血液浄化法の構築につながることが期待されます。

Language：English  

The circadian rhythm, which regulates various body functions, is transcriptionally controlled by a series of clock gene clusters. The clock genes are related to the pathology of various kinds of diseases, which in turn, is related to aging. Aging in humans is a worldwide problem; it induces sleep disorders and disruption of the circadian rhythm. It also decreases ocular vision and appetite and weakens the synchronization of clock genes by light and food. Therefore, a simple method for the synchronization of clock genes in the body is required. In this study, the influence of microcurrent stimulation (MCS) on the circadian machinery in wild-type (WT) and Clock mutant (Clk/Clk) mice was investigated. MCS induced Per1 mRNA expression in cultured mouse astrocytes; cAMP response element (CRE) in the Per1 mouse promoter was found to be important for the induction of Per1 mRNA. In addition, MCS increased the Per1 mRNA levels in mouse livers and caused the phase advance of the Per1 expression rhythm. The protein expression rhythm of phosphor-cAMP response element-binding protein (pCREB) was altered and the phase of expression of pCREB protein advanced. Finally, the influence of MCS on the locomotor activity rhythm in WT and Clk/Clk mice was investigated. MCS caused the phase advance of the locomotor activity rhythm in WT and Clk/Clk mice. The results of this study indicate that MCS activated the clock machinery in mice; MCS may thus improve the quality of new treatment modalities in the future.

DOI： 10.1016/j.bbrc.2019.02.022.

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Although malignant phenotypes of triple-negative breast cancer (TNBC) are subject to circadian alterations, the role of cancer stem cells (CSC) in defining this circadian change remains unclear. CSC are often characterized by high aldehyde dehydrogenase (ALDH) activity, which is associated with the malignancy of cancer cells and is used for identification and isolation of CSC. Here, we show that the population of ALDH-positive cells in a mouse 4T1 breast tumor model exhibits pronounced circadian alterations. Alterations in the number of ALDHpositive cells were generated by time-dependent increases and decreases in the expression of Aldh3a1. Importantly, circadian clock genes were rhythmically expressed in ALDH-negative cells, but not in ALDH-positive cells. Circadian expression of Aldh3a1 in ALDH-positive cells was dependent on the timedependent release of Wingless-type mmtv integration site family 10a (WNT10a) from ALDH-negative cells. Furthermore, antitumor and antimetastatic effects of ALDH inhibitor N,N-diethylaminobenzaldehyde were enhanced by administration at the time of day when ALDH activity was increased in 4T1 tumor cells. Our findings reveal a new role for the circadian clock within the tumor microenvironment in regulating the circadian dynamics of CSC. These results should enable the development of novel therapeutic strategies for treatment of TNBC with ALDH inhibitors. Significance: This seminal report reveals that circadian dynamics of CSC are regulated by the tumor microenvironment and provides a proof of principle of its implication for chronotherapy in TNBC.

DOI： 10.1158/0008-5472.CAN-17-4034

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DOI： 10.1016/j.jbc.2025.108544

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DOI： 10.1523/JNEUROSCI.1969-24.2025

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Language：English   Publisher：Biochemical and Biophysical Research Communications  

Circadian rhythm alterations are related to the onset and severity of various diseases. The expression of the dopamine receptor D3 (DRD3) is regulated by clock genes, and DRD3 functional abnormalities are linked to various neurological diseases. However, the relationship between DRD3 function and circadian machinery is unclear. Here, we demonstrate the influence of DRD3 on the circadian machinery. Although the expression of DRD3 in mouse suprachiasmatic nucleus (SCN) did not show a circadian rhythm, the expression of Per1 mRNA was altered in the SCN of Drd3 knockout (Drd3−/−) mice compared to that in wild-type (WT) mice. These differences were caused by the upregulation of the DRD3–extracellular signal–regulated kinase–cAMP response element binding protein (DRD3–ERK–CREB) signaling pathway in cultured cells and SCN. In addition, Drd3−/− mice demonstrated increased period length of locomotor activity than WT mice only under constant dark conditions. Expression of clock genes in the liver, which does not express DRD3, was affected by the loss of DRD3 only under constant dark conditions, similar to that in the SCN. These results suggest that DRD3 expressed in the SCN regulates the central clock via endogenous ligands and affects peripheral organs. This may provide new evidence to unravel the relationship between dopamine neurotransmission and the circadian clock, which has not yet been fully elucidated.

DOI： 10.1016/j.bbrc.2025.151470

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DOI： 10.1021/acs.jmedchem.2c01081.

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Publisher：医歯薬出版  

DOI： 10.32118/ayu28102169

CiNii Research

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

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

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The expression levels of many cell-surface proteins vary with the time of day. Glycoprotein 2 (Gp2), specifically expressed on the apical surface of M cells in Peyer's patches, functions as a transcytotic receptor for mucosal antigens. We report that cAMP response element-binding protein (CREB) regulates the transcription of the Gp2 gene, thereby generating the circadian change in its expression in mouse Peyer's patches. The transcytotic receptor activity of Gp2 was increased during the dark phase when the Gp2 protein abundance increased. Rhythmic expression of clock gene mRNA was observed in mouse Peyer's patches, and expression levels of Gp2 mRNA also exhibited circadian oscillation, with peak levels during the early dark phase. The promoter region of the mouse Gp2 gene contains several cAMP response elements (CREs). Chromatin immunoprecipitation assays revealed that CREB bound to the CREs in the Gp2 gene in Peyer's patches. Forskolin, which promotes CREB phosphorylation, increased the transcription of the Gp2 gene in Peyer's patches. As phosphorylation of CREB protein was increased when Gp2 gene transcription was activated, CREB may regulate the rhythmic expression of Gp2 mRNA in Peyer's patches. These findings suggest that intestinal immunity is controlled by the circadian clock system.

DOI： 10.1111/gtc.12758

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Detection of metabolic activity in living cells facilitates the understanding of the cell mechanism. Here, we report a fluorescent probe that can detect fatty acid beta oxidation (FAO) in living cells. This probe is metabolically degraded by the sequential enzyme reactions of FAO and can visualize the FAO activity with turn-on fluorescence.

DOI： 10.1039/c9cc09993j

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P-glycoprotein (P-gp/ABCB1) is an ATP-binding cassette drug efflux transporter expressed in a variety of tissues that affects the pharmacokinetic disposition of many drugs. Although several studies have reported gender-dependent differences in the expression of P-gp, the role of sex hormones in regulating the expression of P-gp and its transport activity has not been well understood. In this study, we demonstrated that 17β-estradiol has the ability to induce the expression of P-pg in mouse kidneys and cultured human renal proximal tubular epithelial cells. After intravenous injection of a typical P-gp substrate, digoxin, renal clearance in female mice was approximately 2-fold higher than that in male mice. The expression of murine P-gp and its mRNA (Abcb1a and Abcb1b) were also higher in female mice than in male mice. The expression of P-gp in cultured renal tissues prepared from female and male mice was significantly increased by 17β-estradiol, but not testosterone. Similar 17β-estradiol-induced expression of P-gp was also detected in cultured human tubular epithelial cells, accompanied by the enhancement of its transport activity of digoxin. The present findings suggest the contribution of estradiol to female-predominant expression of P-gp in renal cells, which is associated with sex-related disparities in the renal elimination of digoxin.

DOI： 10.1016/j.bbrc.2019.09.021

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In mammals, the circadian pacemaker resides in the paired suprachiasmatic nuclei (SCN) and influences a multitude of biological processes, including the sleep–wake rhythm. Circadian rhythms regulate diverse physiologic processes, including homeostatic functions of steroid hormones and their receptors. Perturbation of these rhythms is associated with pathogenic conditions such as cancer, metabolic syndrome, cardiovascular disease, sleep disorder and depression. Clock genes ultimately control a vast array of circadian rhythms involved in physiology and behavior. They regulate several diseases described above. Chronotherapy is especially relevant when the risk and/or intensity of symptoms of a disease vary predictably over time. The effectiveness and toxicity of several drugs vary depending on the dosing time. Such chronopharmacological phenomena are influenced by not only the pharmacodynamics but also the pharmacokinetics of a medication. The underlying mechanisms are associated with the 24-h rhythms of biochemical, physiological, and behavioral processes under the control of the circadian clock. Identifying a rhythmic marker based on the molecular clock for choosing dosing time can lead to the progress and diffusion of chronopharmacotherapy. To monitor the rhythmic markers such as clock genes, it might be useful to choose the most appropriate time of a day for the administration of a drug, to increase its therapeutic effects and/or reduce its side effects. On the contrary, several drugs affect the molecular clock and alter the 24-h rhythms of various processes. Alterations in rhythmicity are sometimes associated with therapeutic effects, or it might lead to illness and altered homeostatic regulation. Furthermore, to produce new rhythmicity by manipulating the molecular clock of organs by rhythmic administration of drugs at altered feeding schedules appears to lead to a new concept of chronopharmacotherapy. An approach to increase the efficiency of pharmacotherapy is administering drugs at times when they are best tolerated. From the perspective of pharmaceutics, the application of biological rhythm to pharmacotherapy can be accomplished by the appropriate timing of administration of conventionally formulated tablets and capsules, and also by the use of special drug-delivery system to synchronize drug concentrations to the rhythms in disease activity. New drugs targeting the molecular clock are being developed to manage diseases in human. For instance, novel molecular mechanisms that mediate renal dysfunction in mice with chronic kidney disease (CKD) have been identified by examining the relationship between the circadian clock and CKD aggravation. The inhibition of cell cycle regulatory factor ameliorated renal inflammation in a mouse model of CKD. A novel inhibitor of cell cycle regulatory factor has been identified, supporting the potential utility of cell cycle regulatory factor inhibition in the treatment of CKD. Although malignant phenotypes of triple-negative breast cancer are subject to circadian alterations, the role of cancer stem cells (CSCs) in defining this circadian change remains unclear. The effectiveness of anticancer drugs varies with the circadian dynamics of CSCs, which are regulated by the tumor microenvironmental factors. The finding reveals that the circadian dynamics of CSCs are regulated by the tumor microenvironment and provides a proof of principle of its implication for chronotherapy against triple-negative breast cancer. Therefore, we present an overview of the dosing-time-dependent alterations in therapeutic outcome and safety of a drug and the regulatory system of biological rhythm from the perspective of clock genes and the possibility of pharmacotherapy based on the molecular clock.

DOI： 10.1016/j.pharmthera.2019.05.018

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

Background and Purpose: Doxorubicin is a highly effective anticancer agent but eventually induces cardiotoxicity associated with increased production of ROS. We previously reported that a pathological protein interaction between TRPC3 channels and NADPH oxidase 2 (Nox2) contributed to doxorubicin-induced cardiac atrophy in mice. Here we have investigated the effects of ibudilast, a drug already approved for clinical use and known to block doxorubicin-induced cytotoxicity, on the TRPC3-Nox2 complex. We specifically sought evidence that this drug attenuated doxorubicin-induced systemic tissue wasting in mice. Experimental Approach: We used the RAW264.7 macrophage cell line to screen 1,271 clinically approved chemical compounds, evaluating functional interactions between TRPC3 channels and Nox2, by measuring Nox2 protein stability and ROS production, with and without exposure to doxorubicin. In male C57BL/6 mice, samples of cardiac and gastrocnemius muscle were taken and analysed with morphometric, immunohistochemical, RT-PCR and western blot methods. In the passive smoking model, cells were exposed to DMEM containing cigarette sidestream smoke. Key Results: Ibudilast, an anti-asthmatic drug, attenuated ROS-mediated muscle toxicity induced by doxorubicin treatment or passive smoking, by inhibiting the functional interactions between TRPC3 channels and Nox2, without reducing TRPC3 channel activity. Conclusions and Implications: These results indicate a common mechanism underlying induction of systemic tissue wasting by doxorubicin. They also suggest that ibudilast could be repurposed to prevent muscle toxicity caused by anticancer drugs or passive smoking.

DOI： 10.1111/bph.14777

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DOI： 10.1038/s41589-018-0204-3.

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Growth of cancer cells is more highly dependent on various types of amino acids than that of normal cells, and thus prevention of amino acid requirement has been recognized as strategies for cancer therapies. In this study, we found that deprivation of cysteine (Cys) in culturing media prevented the growth of various types of human cancer cell lines. Cys is easily converted to cystine (Cys–Cys) in media and uptaken into cells by cystine/glutamate transporter (xCT). The incorporated Cys–Cys is decomposed into Cys, and used for synthesis of glutathione that suppresses reactive oxygen species-induced cell damage. Therefore, we examined whether a selective xCT inhibitor erastin prevented the growth of human cancer cell lines. As a result, erastin significantly prevented the proliferation of various types of human cancer cells. Among them, MDA-MB-231 breast cancer cells were identified as the most erastin-sensitive cells. To investigate the ability of erastin to prevent growth of tumor in mice, MDA-MB-231 breast cancer cells were implanted into BALB/c nude female mice kept under standardized light/dark cycle conditions. The growth of tumor implanted in mice was significantly suppressed by administration of erastin during the light phase, whereas its administration during the dark phase failed to suppress the tumor growth. The dosing time-dependency of erastin-induced cystine/ cysteine deprivation was closely related to that of its anti-tumor effects. Our present findings suggest that the anti-tumor efficacy of erastin in tumor-bearing mice is improved by optimizing the dosing schedule.

DOI： 10.1248/bpb.b19-00546

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Disruption of circadian rhythms has been implicated in an increased risk for cancer development. The Period2 (Per2) gene encodes one of the major components of the mammalian circadian clock, which plays a key role in controlling the circadian rhythms in physiology and behavior. PER2 has also been reported to suppress the malignant transformation of cells, but its role in the regulation of cancer susceptibility to chemotherapeutic drugs remains unclear. In this study, we found that oncogene-transformed embryonic fibroblasts prepared from Per2-mutant (Per2^m/m) mice, which are susceptible to both spontaneous and radiation-induced tumorigenesis, were resistant against common chemotherapeutic drugs and that this resistance is associated with up-regulation of the aldehyde dehydrogenase 3a1 (Aldh3a1) gene. Co-expression of the oncogenes H-ras^V12 and SV40 large T-antigen induced malignant transformation of both WT and Per2^m/m cells, but the cytotoxic effects of the chemotherapeutic agents methotrexate, gemcitabine, etoposide, vincristine, and oxaliplatin were significantly alleviated in the oncogene-transformed Per2^m/m cells. Although introduction of the two oncogenes increased the expression of Aldh3a1 in both WT and Per2^m/m cells, the ALDH3A1 protein levels in the Per2^m/m cells were 7-fold higher than in WT cells. The elevated ALDH3A1 levels in the oncogene-transformed Per2^m/m cells were sufficient to prevent chemotherapeutic drug–induced accumulation of reactive oxygen species. Consequently, shRNA-mediated suppression of Aldh3a1 expression relieved the chemoresistance of the Per2^m/m cells. These results suggest a role for mutated PER2 in the development of multiple drug resistance and may inform therapeutic strategies for cancer management.

DOI： 10.1074/jbc.RA118.004942

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A number of diverse cell-surface proteins are anchored to the cytoskeleton via scaffold proteins. Na⁺/H⁺ exchanger regulatory factor-1 (NHERF1), encoded by the Slc9a3r1 gene, functions as a scaffold protein, which is implicated in the regulation of membrane expression of various cell-surface proteins. Here, we demonstrate that the circadian clock component PERIOD2 (PER2) modulates transcription of the mouse Slc9a3r1 gene, generating diurnal accumulation of NHERF1 in the mouse liver. Basal expression of Slc9a3r1 was dependent on transcriptional activation by p65/p50. PER2 bound to p65 protein and prevented p65/p50-mediated transactivation of Slc9a3r1. The time-dependent interaction between PER2 and p65 underlay diurnal oscillation in the hepatic expression of Slc9a3r1/NHERF1. The results of immunoprecipitation experiments and liquid chromatography-mass spectrometry analysis of mouse liver revealed that NHERF1 time-dependently interacted with fatty acid transport protein-5 (FATP5). Temporary accumulation of NHERF1 protein stabilized plasmalemmal localization of FATP5, thereby enhancing hepatic uptake of fatty acids at certain times of the day. Our results suggest an unacknowledged role for PER2 in regulating the diurnal expression of NHERF1 in mouse liver. This machinery also contributed to diurnal changes in the ability of hepatic cells to uptake fatty acids.

DOI： 10.1038/s41598-018-27280-w

Language：English  

The circadian rhythm, which regulates various body functions, is transcriptionally controlled by a series of clock gene clusters. The clock genes are related to the pathology of various kinds of diseases. Although there is evidence of serious sleep disorders in patients with chronic hepatitis, the liver regeneration mechanism under chronic hepatitis conditions and its association with the clock genes are not clear. In this study, the influence of the circadian locomotor output cycles kaput (CLOCK), which is one of the clock genes, on a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced hepatitis animal model was investigated. The appearance of potential hepatic stem-like cells (epithelial cell adhesion molecule [EpCAM]-positive cells) is an initial critical step in liver regeneration during chronic inflammation. The results showed a considerable number of hepatic EpCAM-positive cells in the wild-type (WT) mice 1 week after the DDC feeding. However, the number of EpCAM-positive cells in the Clock-mutant (Clk/Clk) mice decreased, and their hepatitis was worse compared with the WT mice. In addition, the expression of Epcam mRNA, which is a functional marker of potential hepatic stem-like cells, was controlled by LEF1, which was regulated by CLOCK. The results of this study will facilitate the elucidation of the liver regeneration mechanisms, including those at the molecular level, and may assist in the development of new treatment modalities in the future.

DOI： 10.1016/j.bbrc.2018.06.117

Language：English  

The intensity of pain in diabetic neuropathy varies in a circadian time-dependent manner. It is well known that diabetes has two distinct types, which are differentiated based on the cause of the disease. Previous studies have yet to compare the circadian properties of the pain intensity of diabetic neuropathy between type I and type II diabetes. In this study, we demonstrated that the pain intensity of diabetic peripheral neuropathy in a db/db mouse model of type II diabetes showed a significant diurnal oscillation, but such time-dependent oscillation was not detected in a streptozotocin (STZ)-induced type I diabetic mouse model. The polyol pathway-induced accumulation of sorbitol in peripheral nerve cells suppresses Na⁺/K⁺-ATPase activity, which is associated with the intensity of pain in diabetic neuropathy. In db/db mice, this accumulation of sorbitol in peripheral nerve cells showed significant diurnal oscillation. In addition, pain intensity and Na⁺/K⁺-ATPase activity were decreased at the peak time of sorbitol accumulation in these mice. Although STZ-induced diabetic mice also showed sorbitol accumulation and Na⁺/K⁺-ATPase dysfunction, these measures did not oscillate in a time-dependent manner. These findings reveal differences in the circadian properties of pain hypersensitivity in mouse models of type I and type II diabetes, and also provide ideas for developing novel approaches to the management of diabetic neuropathy.

DOI： 10.1016/j.bbrc.2018.05.209

Language：English  

Proliferation of acute lymphoblastic leukemic cells is nutritionally dependent on the external supply of asparagine. L-asparaginase, an enzyme hydrolyzing L-asparagine in blood, is used for treatment of acute lymphoblastic leukemic and other related blood cancers. Although previous studies demonstrated that L-asparaginase suppresses the proliferation of cultured solid tumor cells, it remains unclear whether this enzyme prevents the growth of solid tumors in vivo. In this study, we demonstrated the importance of optimizing dosing schedules for the anti-tumor activity of L-asparaginase in 4T1 breast tumor-bearing mice. Cultures of several types of murine solid tumor cells were dependent on the external supply of asparagine. Among them, we selected murine 4T1 breast cancer cells and implanted them into BALB/c female mice kept under standardized light/dark cycle conditions. The growth of 4T1 tumor cells implanted in mice was significantly suppressed by intravenous administration of L-asparaginase during the light phase, whereas its administration during the dark phase failed to show significant anti-tumor activity. Decreases in plasma asparagine levels due to the administration of L-asparaginase were closely related to the dosing time-dependency of its anti-tumor effects. These results suggest that the anti-tumor efficacy of L-asparaginase in breast tumor-bearing mice is improved by optimizing the dosing schedule.

DOI： 10.1016/j.jphs.2018.01.008

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

One approach to increasing pharmacotherapy effects is administering drugs at times of day when they are most effective and/or best tolerated. Circadian variation in expression of pharmacokinetics- and pharmacodynamics-related genes was shown to contribute to dosing time-dependent differences in therapeutic effects of small molecule drugs. However, influence of dosing time of day on effects of high molecular weight formulations, such as drugs encapsulated in liposomes, has not been studied in detail. This study demonstrates that blood pressure rhythm affects dosing time-dependent variation in effects of high molecular weight formulations. Systolic blood pressure in sarcoma 180-bearing mice showed significant 24-h oscillation. Intratumoral accumulation of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA), an indicator of tumor vascular permeability, varied with dosing time of day, matching phases of blood pressure circadian rhythm. Furthermore, intratumoral accumulation of liposome-encapsulated oxaliplatin (Lipo-L-OHP) increased with increases in systolic blood pressure. Our findings suggest that circadian blood pressure oscillations may be an important factor to consider in dosing strategies for macromolecular drugs and liposomes in cancer therapy.

DOI： 10.1016/j.bbrc.2017.11.162

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

Purpose: Clock genes are components of the molecular clock. Their malfunction is thought to increase the risk of numerous diseases, including cancer. Vascular endothelial growth factor (VEGF) has a pivotal role in angiogenesis, and its expression levels are controlled by clock genes in tumor cells. Ophthalmic diseases such as age-related macular degeneration, proliferative diabetic retinopathy, and neovascular glaucoma are also associated with abnormal angiogenesis followed by upregulation of VEGF in the eye. In the present study, we aimed to uncover the relationship between clock genes and VEGF in the eye. Study design: Laboratory investigation Methods: Oxygen-induced retinopathy (OIR) mice were prepared to mimic hypoxic conditions in the eye. Deferoxamine (DFO) was used to mimic hypoxic conditions in human Müller cell line MIO-M1 cells. Expression levels of mRNA and protein were quantified by quantitative reverse transcription polymerase chain reaction and Western blot analysis, respectively. Results: In the retinas of OIR mice, the expression levels of Vegf and the clock gene Dec2 increased transiently, and their temporal profiles were correlated. Knockdown of DEC2 resulted in a significant (26.7%) reduction of VEGF expression in MIO-M1 cells under hypoxia-mimicking conditions induced by DFO (P <.05). Levels of HIF1α protein were also reduced significantly, by 60.2%, in MIO-M1 cells treated with siRNA against the DEC2 gene (P <.05). Moreover, HIF1α levels showed a significant (2.5-fold) increase in MIO-M1 cells overexpressing DEC2 (P <.05). Conclusion: DEC2 could upregulate retinal VEGF gene expression through modulation of HIF1α levels under hypoxic conditions.

DOI： 10.1007/s10384-018-0622-5

Language：English  

The chronic disruption of circadian rhythms has been implicated in the risk of cancer development in humans and laboratory animals. The gene product CLOCK is a core molecular component of the circadian oscillator, so that mice with a mutated Clock gene (Clk/Clk) exhibit abnormal rhythms in various physiological processes. However, we demonstrated here that Clk/Clk mice resisted chemical carcinogen-induced tumorigenesis by suppressing epidermal growth factor (EGF) receptor-mediated proliferation signals. The repetitive application of 7,12-dimethylbenz[α]anthracene (DMBA) to skin on the back resulted in the significant development of tumors in wild-type mice, whereas chemically-induced tumorigenesis was alleviated in Clk/Clk mice. Although the degree of DMBA-induced DNA damage was not significantly different between wild-type and Clk/Clk mice, EGF receptor-mediated Ras activation was not detected in DMBA-treated Clk/Clk mice. Genetic and biochemical experiments revealed that the suppression of EGF receptor-mediated signal transduction in DMBA-treated Clk/Clk mice was associated with the expression of the cellular senescence factor p16INK4a. These results suggest an uncovered role for CLOCK in the development of chemical carcinogen-induced primary tumors and offers new preventive strategies.

DOI： 10.1038/s41598-017-10599-1

Language：English  

Clock genes encoding transcription factors that regulate circadian rhythms may inform chronomodulated chemotherapy, where time-dependent dose alterations might affect drug efficacy and reduce side effects. For example, inhibiting the essential cystine transporter xCT with sulfasalazine induces growth arrest in cancer cells. Although the anticancer effects of sulfasalazine have been studied extensively, its effects on transcriptional control of xCT expression have not been studied. Here, we show that sulfasalazine administration during the period of increased xCT expression improves its anticancer effects and that the Clock gene itself induces xCT expression and regulates its circadian rhythm. Our findings highlight the clinical potential of chronomodulated chemotherapy and the importance of xCT-mediated transcriptional regulation in the utility of such strategies.

DOI： 10.1158/0008-5472.CAN-17-0720

Language：English  

Xanthine oxidase (XOD), also known as xanthine dehydrogenase, is a rate-limiting enzyme in purine nucleotide degradation, which produces uric acid. Uric acid concentrations in the blood and liver exhibit circadian oscillations in both humans and rodents; however, the underlying mechanisms remain unclear. Here, we demonstrate that XOD expression and enzymatic activity exhibit circadian oscillations in the mouse liver. We found that the orphan nuclear receptor peroxisome proliferator-activated receptor-α (PPARα) transcriptionally activated the mouse XOD gene and that bile acids suppressed XOD transactivation. The synthesis of bile acids is known to be under the control of the circadian clock, and we observed that the time-dependent accumulation of bile acids in hepatic cells interfered with the recruitment of the co-transcriptional activator p300 to PPARα, thereby repressing XOD expression. This time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the hepatic expression of XOD, which, in turn, led to circadian alterations in uric acid production. Finally, we also demonstrated that the anti-hyperuricemic effect of the XOD inhibitor febuxostat was enhanced by administering it at the time of day before hepatic XOD activity increased. These results suggest an underlying mechanism for the circadian alterations in uric acid production and also underscore the importance of selecting an appropriate time of day for administering XOD inhibitors.

DOI： 10.1074/jbc.M117.791285

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

Clock genes encoding transcription factors that regulate circadian rhythms may inform chronomodulated chemotherapy, where time-dependent dose alterations might affect drug efficacy and reduce side effects. For example, inhibiting the essential cystine transporter xCT with sulfasalazine induces growth arrest in cancer cells. Although the anticancer effects of sulfasalazine have been studied extensively, its effects on transcriptional control of xCT expression have not been studied. Here, we show that sulfasalazine administration during the period of increased xCT expression improves its anticancer effects and that the Clock gene itself induces xCT expression and regulates its circadian rhythm. Our findings highlight the clinical potential of chronomodulated chemotherapy and the importance of xCT-mediated transcriptional regulation in the utility of such strategies.

DOI： 10.1158/0008-5472.CAN-17-0720

Language：English  

Maintenance of circadian rhythms is essential to many aspects of human health, including metabolism and neurological and psychiatric well-being. Chronic disruption of circadian clock function is implicated in increasing the risk of metabolic syndrome, cardiovascular events and development of cancers. However, there are little approaches to reinforce the function of circadian clock for prevention of these diseases. Essence of Chicken (EC) is a nutritional supplement that is traditionally made by extracting water soluble substances derived from cooking the whole chicken. In this study, we found that dietary supplementation with EC enhanced circadian oscillation of glucocorticoid secretion in mice, and this was accompanied by enhancement of circadian oscillation in the adrenal expression of steroidogenic acute regulatory (StAR) protein that mediates the rate-limiting step of glucocorticoid synthesis. Furthermore, EC facilitated re-entrainment of behavioral rhythm in mice when phase of the light–dark cycle was suddenly advanced. These results suggest that intake of EC has enhancement effect on circadian clock function in mice, which may contribute to sustain health and also offer new preventive strategies against circadian-related diseases.

DOI： 10.1016/j.jphs.2017.07.004

Language：English  

DOI： 10.2745/dds.32.72

Language：English  

Vancomycin (VCM) is a first-line antibiotic for serious infections caused by methicillin-resistant Staphylococcus aureus. However, nephrotoxicity is one of the most complaint in VCM therapy. We previously reported that VCM induced apoptosis in a porcine proximal tubular epithelial cell line (LLC-PK1), in which mitochondrial complex I may generate superoxide, leading to cell death. In the present study, VCM caused production of mitochondrial reactive oxygen species and peroxidation of the mitochondrial phospholipid cardiolipin that was reversed by administration of the mitochondrial uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). FCCP also significantly suppressed VCM-induced depolarization of the mitochondrial membrane and apoptosis. Moreover, the lipophilic antioxidant vitamin E and a mitochondria-targeted antioxidant, mitoTEMPO, also significantly suppressed VCM-induced depolarization of mitochondrial membrane and apoptosis, whereas vitamin C, n-acetyl cysteine, or glutathione did not provide significant protection. These findings suggest that peroxidation of the mitochondrial membrane cardiolipin mediated the VCM-induced production of intracellular reactive oxygen species and initiation of apoptosis in LLC-PK1 cells. Furthermore, regulation of mitochondrial function using a mitochondria-targeted antioxidant, such as mitoTEMPO, may constitute a potential strategy for mitigation of VCM-induced proximal tubular epithelial cell injury.

DOI： 10.1016/j.ejphar.2017.02.025

Language：English  

Xanthine oxidase (XOD), also known as xanthine dehydrogenase, is a rate-limiting enzyme in purine nucleotide degradation, which produces uric acid. Uric acid concentrations in the blood and liver exhibit circadian oscillations in both humans and rodents; however, the underlying mechanisms remain unclear. Here, we demonstrate that XOD expression and enzymatic activity exhibit circadian oscillations in the mouse liver. We found that the orphan nuclear receptor peroxisome proliferator-activated receptor-α (PPARα) transcriptionally activated the mouse XOD gene and that bile acids suppressed XOD transactivation. The synthesis of bile acids is known to be under the control of the circadian clock, and we observed that the time-dependent accumulation of bile acids in hepatic cells interfered with the recruitment of the co-transcriptional activator p300 to PPARα, thereby repressing XOD expression. This time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the hepatic expression of XOD, which, in turn, led to circadian alterations in uric acid production. Finally, we also demonstrated that the anti-hyperuricemic effect of the XOD inhibitor febuxostat was enhanced by administering it at the time of day before hepatic XOD activity increased. These results suggest an underlying mechanism for the circadian alterations in uric acid production and also underscore the importance of selecting an appropriate time of day for administering XOD inhibitors.

DOI： 10.1074/jbc.M117.791285

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

DOI： 10.2745/dds.32.72

Language：English  

Vancomycin (VCM) is a first-line antibiotic for serious infections caused by methicillin-resistant Staphylococcus aureus. However, nephrotoxicity is one of the most complaint in VCM therapy. We previously reported that VCM induced apoptosis in a porcine proximal tubular epithelial cell line (LLC-PK1), in which mitochondrial complex I may generate superoxide, leading to cell death. In the present study, VCM caused production of mitochondrial reactive oxygen species and peroxidation of the mitochondrial phospholipid cardiolipin that was reversed by administration of the mitochondrial uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). FCCP also significantly suppressed VCM-induced depolarization of the mitochondrial membrane and apoptosis. Moreover, the lipophilic antioxidant vitamin E and a mitochondria-targeted antioxidant, mitoTEMPO, also significantly suppressed VCM-induced depolarization of mitochondrial membrane and apoptosis, whereas vitamin C, n-acetyl cysteine, or glutathione did not provide significant protection. These findings suggest that peroxidation of the mitochondrial membrane cardiolipin mediated the VCM-induced production of intracellular reactive oxygen species and initiation of apoptosis in LLC-PK1 cells. Furthermore, regulation of mitochondrial function using a mitochondria-targeted antioxidant, such as mitoTEMPO, may constitute a potential strategy for mitigation of VCM-induced proximal tubular epithelial cell injury.

DOI： 10.1016/j.ejphar.2017.02.025

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

Chronic kidney disease (CKD) is a global health problem, and novel therapies to treat CKD are urgently needed. Here, we show that inhibition of G₀/G₁ switch 2 (G0s2) ameliorates renal inflammation in a mouse model of CKD. Renal expression of chemokine (C-C motif) ligand 2 (Ccl2) was increased in response to p65 activation in the kidneys of wild-type 5/6 nephrectomy (5/6Nx) mice. Moreover, 5/6Nx Clk/Clk mice, which carry homozygous mutations in the gene encoding circadian locomotor output cycles kaput (CLOCK), did not exhibit aggravation of apoptosis or induction of F4/80-positive cells. The renal expression of G0s2 in wild-type 5/6Nx mice was important for the transactivation of Ccl2 by p65. These pathologies were ameliorated by G0s2 knockdown. Furthermore, a novel small-molecule inhibitor of G0s2 expression was identified by high-throughput chemical screening, and the inhibitor suppressed renal inflammation in 5/6Nx mice. These findings indicated that G0s2 inhibitors may have applications in the treatment of CKD.

DOI： 10.1016/j.ebiom.2016.10.008

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

Diurnal variations in pain hypersensitivity are common in chronic pain disorders, but the underlying mechanisms are enigmatic. Here, we report that mechanical pain hypersensitivity in sciatic nerve-injured mice shows pronounced diurnal alterations, which critically depend on diurnal variations in glucocorticoids from the adrenal glands. Diurnal enhancement of pain hypersensitivity is mediated by glucocorticoid-induced enhancement of the extracellular release of ATP in the spinal cord, which stimulates purinergic receptors on microglia in the dorsal horn. We identify serum- and glucocorticoid-inducible kinase-1 (SGK-1) as the key molecule responsible for the glucocorticoid-enhanced release of ATP from astrocytes. SGK-1 protein levels in spinal astrocytes are increased in response to glucocorticoid stimuli and enhanced ATP release by opening the pannexin-1 hemichannels. Our findings reveal an unappreciated circadian machinery affecting pain hypersensitivity caused by peripheral nerve injury, thus opening up novel approaches to the management of chronic pain.

DOI： 10.1038/ncomms13102

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

In mammals, circadian rhythms in physiological function are generated by a molecular oscillator driven by transcriptional-translational feedback loop consisting of negative and positive regulators. Disruption of this circadian clock machinery is thought to increase the risk of cancer development, but the potential contributions of each component of circadian clock to oncogenesis have been little explored. Here we reported that negative and positive transcriptional regulators of circadian feedback loop had different roles in oncogene-induced neoplastic transformation. Mouse embryonic fibroblasts prepared from animals deficient in negative circadian clock regulators, Period2 (Per2) or Cryptochrome1/2 (Cry1/2), were prone to transformation induced by co-expression of H-ras^V12 and SV40 large T antigen (SV40LT). In contrast, mouse embryonic fibroblasts prepared from mice deficient in positive circadian clock regulators, Bmal1 or Clock, showed resistance to oncogene-induced transformation. In Per2 mutant and Cry1/2-null cells, the introduction of oncogenes induced expression of ATF4, a potent repressor of cell senescence-associated proteins p16INK4a and p19ARF. Elevated levels of ATF4 were sufficient to suppress expression of these proteins and drive oncogenic transformation. Conversely, in Bmal1-null and Clock mutant cells, the expression of ATF4 was not induced by oncogene introduction, which allowed constitutive expression of p16INK4a and p19ARF triggering cellular senescence. Although genetic ablation of either negative or positive transcriptional regulators of the circadian clock leads to disrupted rhythms in physiological functions, our findings define their different contributions to neoplastic cellular transformation.

DOI： 10.1074/jbc.M115.706481

Language：English  

Chronic kidney disease (CKD) is associated with an increase in serum retinol; however, the underlying mechanisms of this disorder are poorly characterized. Here, we found that the alteration of hepatic metabolism induced the accumulation of serum retinol in 5/6 nephrectomy (5/6Nx) mice. The liver is the major organ responsible for retinol metabolism; accordingly, microarray analysis revealed that the hepatic expression of most CYP genes was changed in 5/6Nx mice. In addition, D-box-binding protein (DBP), which controls the expression of several CYP genes, was significantly decreased in these mice. Cyp3a11 and Cyp26a1, encoding key proteins in retinol metabolism, showed the greatest decrease in expression in 5/6Nx mice, a process mediated by the decreased expression of DBP. Furthermore, an increase of plasma transforming growth factor-β1 (TGF-β1) in 5/6Nx mice led to the decreased expression of the Dbp gene. Consistent with these findings, the alterations of retinol metabolism and renal dysfunction in 5/6Nx mice were ameliorated by administration of an anti-TGF-β1 antibody. We also show that the accumulation of serum retinol induced renal apoptosis in 5/6Nx mice fed a normal diet, whereas renal dysfunction was reduced in mice fed a retinol-free diet. These findings indicate that constitutive Dbp expression plays an important role in mediating hepatic dysfunction under CKD. Thus, the aggravation of renal dysfunction in patients with CKD might be prevented by a recovery of hepatic function, potentially through therapies targeting DBP and retinol.

DOI： 10.1074/jbc.M115.696930

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

Iron is an important biological catalyst and is critical forDNA synthesis during cell proliferation. Cellular iron uptake is enhanced in tumor cells to support increased DNA synthesis. Circadian variations in DNA synthesis and proliferation have been identified in tumor cells, but their relationship with intracellular iron levels is unclear. In this study, we identified a 24-h rhythm in iron regulatory protein 2 (IRP2) levels in colon-26 tumors implanted in mice. Our findings suggest that IRP2 regulates the 24-h rhythm of transferrin receptor 1 (Tfr1) mRNA expression post-transcriptionally, by binding toRNAstem-loop structures known as iron-response elements.Wealso found that Irp2 mRNA transcription is promoted by circadian clock genes, including brain and muscle Arnt-like 1 (BMAL1) and the circadian locomotor output cycles kaput (CLOCK) heterodimer. Moreover, growth in colon-26(^δ19) tumors expressing the clock-mutant protein (CLOCK^δ19) was low compared with that in wild-type colon-26 tumor. The time-dependent variation of cellular iron levels, and the proliferation rate in wild-type colon-26 tumor was decreased by CLOCK^δ19 expression. Our findings suggest that circadian organization contributes to tumor cell proliferation by regulating iron metabolism in the tumor.

DOI： 10.1074/jbc.M115.713412

Language：English  

Bisphosphonates and statins are known to have antitumor activities against different types of cancer cell lines. In the present study, we investigated the antiproliferative effects of the combination of zoledronic acid (ZOL), a bisphophosphonate, and fluvastatin (FLU), a statin, in vitro on two types of human pancreatic cancer cell lines, Mia PaCa-2 and Suit-2. The pancreatic cancer cell lines were treated with ZOL and FLU both individually and in combination to evaluate their antiproliferative effects using WST-8 cell proliferation assay. In this study, we demonstrated a potent synergistic antiproliferative effect of both drugs when used in combination in both cell lines. Moreover, we studied the molecular mechanism behind this synergistic effect, which was inhibited by the addition of the mevalonate pathway products, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Furthermore, we aimed to determine the effect of ZOL and FLU combination on RhoA and Ras guanosine 5′-triphosphate (GTP)-proteins. The combination induced a marked accumulation in RhoA and unprenylated Ras. GGPP and FPP reversed the increase in the amount of both proteins. These results indicated that the combination treatment impaired RhoA and Ras signaling pathway by the inhibition of geranylgeranylation and/or farnesylation. This study provides a potentially effective approach for the treatment of pancreatic cancer using a combination treatment of ZOL and FLU.

DOI： 10.1248/bpb.b15-00746

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

Clock gene regulates the circadian rhythm of various physiological functions. The expression of clock gene has been shown to be attenuated by certain drugs, resulting in a rhythm disorder. Mitomycin C (MMC) is often used in combination with ophthalmic surgery, especially in trabeculectomy, a glaucoma surgical procedure. The purpose of this study was to investigate the influence of MMC on clock gene expression in fibroblasts, the target cells of MMC. Following MMC treatment, Bmal1 mRNA levels was significantly decreased, whereas Dbp, Per1, and Rev-erbα mRNA levels were significantly increased in the mouse fibroblast cell line NIH3T3 cells. Microarray analysis was performed to explore of the gene(s) responsible for MMC-induced alteration of clock gene expression, and identified Nr3c1 gene encoding glucocorticoid receptor (GR) as a candidate. MMC suppressed the induction of Per1 mRNA by dexamethasone (DEX), ligand of GR, in NIH3T3 cells. MMC also modulated the DEX-driven circadian oscillations of Per2::Luciferase bioluminescence in mouse-derived ocular fibroblasts. Our results demonstrate a previously unknown effect of MMC in GR signaling and the circadian clock system. The present findings suggest that MMC combined with trabeculectomy could increase the risk for a local circadian rhythm-disorder at the ocular surface.

DOI： 10.1016/j.bbrc.2015.09.086

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

Recent studies in laboratory rodents have revealed that circadian oscillation in the physiologic functions affecting drug disposition underlies the dosing time-dependent change in pharmacokinetics. However, it is difficult to predict the circadian change in the drug pharmacokinetics in a diurnal human by using the data collected from nocturnal rodents. In this study, we used cynomolgus monkeys, diurnal active animals, to evaluate the relevance of intestinal expression of P-glycoprotein (P-gp) to the dosing time dependency of the pharmacokinetics of its substrates. The rhythmic phases of circadian gene expression in the suprachiasmatic nuclei (the mammalian circadian pacemaker) of cynomolgus monkeys were similar to those reported in nocturnal rodents. On the other hand, the expression of circadian clock genes in the intestinal epithelial cells of monkeys oscillated at opposite phases in rodents. The intestinal expression of P-gp in the small intestine of monkeys was also oscillated in a circadian time-dependent manner. Furthermore, the intestinal absorption of P-gp substrates (quinidine and etoposide) was substantially suppressed by administering the drugs at the times of day when P-gp levels were abundant. By contrast, there was no significant dosing time-dependent difference in the absorption of the non-P-gp substrate (acetaminophen). The oscillation in the intestinal expression of P-gp appears to affect the pharmacokinetics of its substrates. Identification of circadian factors affecting the drug disposition in laboratory monkeys may improve the predictive accuracy of pharmacokinetics in humans.

DOI： 10.1124/mol.114.096735

Language：English  

Patients with diabetes often develop peripheral nerve complications, including numbness and pain in the extremities. Diabetes-induced peripheral neuropathic pain is characterized by hypersensitivity to innocuous stimuli, known as tactile allodynia. Pregabalin (PGN) is currently used to treat diabetes-induced peripheral neuropathy and alleviates allodynia. In the present study, we demonstrated that the antiallodynic effect of PGN on diabetic mice was modulated by circadian changes in its intestinal absorption. A single intraperitoneal administration of 200 mg/kg streptozotocin (STZ) to mice induced type I diabetic pathologic changes that were accompanied by tactile allodynia. The intensity of tactile allodynia in STZ-induced diabetic mice was alleviated by the oral administration of PGN; however, the antiallodynic effect varied according to its dosing time. The analgesic effect of PGN was enhanced by its administration at the times of day when its intestinal absorption was accelerated. Organic cation transporter novel type 1 (Octn1) mediated the uptake of PGN into intestinal epithelial cells. The expression of Octn1 in the small intestine of STZ-induced diabetic mice oscillated in a circadian time-dependent manner. This oscillation in Octn1 appeared to cause the time of day-dependent changes in the intestinal absorption of PGN. Similar dosing time dependencies of the antiallodynic effect of PGN and oscillation in Octn1 expression were also detected in type II diabetic db/db mice. These results suggested that the dosing time-dependent differences in the analgesic effect of PGN were attributable to circadian oscillations in the intestinal expression of Octn1 and also that optimizing its dosing schedule may assist in achieving rational pharmacotherapy for diabetes-induced peripheral neuropathic pain.

DOI： 10.1124/jpet.115.223891

Language：English  

In addition to their digestive actions, bile acids modulate gene expression by altering the activity of peroxisome proliferator-activated receptor-α (PPARα). The modulatory effects of bile acids have been shown to affect the expression of genes responsible for lipid metabolism as well as membrane transporters. Bile acids are secreted in response to food intake and accumulate in intestinal epithelial cells. In the present study, we identified soluble carrier protein family 22 member 4 (Slc22a4), encoding organic cation transporter novel type-1 (Octn1), as a PPARa-regulated gene and its intestinal expression exhibited circadian oscillations in a bile acid-dependent manner. Nocturnally active mice mainly consumed their food around the early dark phase, during which bile acids accumulated in intestinal epithelial cells. PPARa activated the intestinal expression of Slc22a4 mRNA during the light period, and protein levels of Octn1 peaked before the start of the dark phase. The bile acids that accumulated in intestinal epithelial cells suppressed the PPARα-mediated transactivation of Slc22a4 in the dark phase. The time-dependent suppression of PPARα-mediated transactivation by bile acids regulated oscillations in the intestinal expression of Octn1/Slc22a4 during the daily feeding cycle. The results of a pharmacokinetic analysis also revealed that oscillations in the expression of Octn1 caused dosing time-dependent differences in the intestinal absorption of gabapentin (2-[1-(aminomethyl)cyclohexyl]acetic acid). These results suggest a molecular clock-independent mechanism by which bile acid-regulated PPARα activity governs the circadian expression of intestinal organic cation transporters. This mechanism could also account for interindividual variations in the pharmacokinetics of drugs that are substrates of Octn1.

DOI： 10.1124/mol.114.094979

Language：English  

Aquaporin 3 (AQP3) is located in the basal layer of the epidermis and regulates biological functions of skin such as water content and trans-epidermal water loss. A recent study showed that the biological function of skin exhibits a 24-hour rhythm, but the molecular mechanism of the variation remains poorly understood. Here we show that mice mutated in the core clock component CLOCK (Clk/Clk) show decreased stratum corneum hydration. An extensive search for the underlying cause led us to identify AQP3 as a new regulator to control the 24-hour variation in biological functions of skin. In mouse epidermis of wild-type mice, mAqp3 exhibits circadian rhythms; however, these are significantly decreased in Clk/Clk. Luciferase reporter gene analysis revealed that transcription of mAqp3 is activated by D-site-binding protein, a clock gene. A human homolog, hAQP3, also exhibited significant oscillation in human keratinocyte (HaCaT) cells synchronized with medium containing 50&#37; serum, and this rhythm was regulated by the endogenous CLOCK/BMAL1 heterodimer. These data indicate that although the molecular mechanisms underlying the rhythmic expression of mAqp3 and hAQP3 are different, clock genes are involved in time-dependent skin hydration. Our current findings provide a molecular link between the circadian clock and AQP3 function in mouse dorsal skin and HaCaT cells.

DOI： 10.1038/jid.2014.13

Language：English  

Cisplatin, cis-diamminedichloro-platinum (CDDP), is a widely used anticancer agent, the clinical applications of which have been limited by severe nephrotoxicity. Although dosing time-dependent differences in CDDP-induced nephrotoxicity have been reported in both humans and laboratory animals, the underlying mechanism remains unknown. In the present study, we investigated the molecular mechanism for the dosing-time dependency of the nephrotoxic effect of CDDP in mice. CDDP-induced nephrotoxicity was significantly attenuated by injecting CDDP at times of the day when its renal clearance was enhanced. The dosing-time dependency of the nephrotoxic effect was parallel to that of CDDP incorporation into renal DNA. Two types of transporters, organic cation transporter 2 (OCT2, encoded by Slc22a2) and multidrug and toxin extrusion 1 (MATE1, encoded by Slc47a1), are responsible for the renal excretion of CDDP. The expression of OCT2, but not MATE1, exhibited a significant time-dependent oscillation in the kidneys of mice. The circadian expression of OCT2 was closely related to the dosing-time dependency of CDDP incorporation into renal DNA. Molecular components of the circadian clock regulated the renal expression of Slc22a2 mRNA by mediating peroxisome proliferator-activated receptor-a, which resulted in rhythmic oscillations in OCT2 protein levels. These findings indicate a clock-regulated mechanism of dosing time-dependent changes in CDDP-induced nephrotoxicity and also suggest a molecular link between the circadian clock and renal xenobiotic excretion.

DOI： 10.1124/mol.113.089805

Language：English  

Circadian clock systems regulate many biologic functions, including cell division and hormone secretion in mammals. In this study, we explored the effects of circadian control on the pivot cell growth regulatory mTOR, the activity of which is deregulated in tumor cells compared with normal cells. Specifically, we investigated whether the antitumor effect of an mTOR inhibitor could be improved by changing its dosing schedule in RenCa tumorbearing mice. Active, phosphorylated mTOR displayed a 24-hour rhythm, and levels of total mTOR protein (but not mRNA) also showed a circadian rhythm in RenCa tumor masses. Through investigations of the oscillation mechanism for mTOR expression, we identified the ubiquitination factor Fbxw7 as an mTOR regulator that oscillated in its expression in a manner opposite from mTOR. Fbxw7 transcription was regulated by the circadian regulator D-site-binding protein. Notably, administration of the mTOR inhibitor everolimus during periods of elevated mTOR improved survival in tumor-bearing mice. Our findings demonstrate that the circadian oscillation of mTOR activity is regulated by circadian clock systems, which influence the antitumor effect of mTOR inhibitors.

DOI： 10.1158/0008-5472.CAN-12-3241

Language：English  

Digested proteins are mainly absorbed as small peptides composed of two or three amino acids. The intestinal absorption of small peptides is mediated via only one transport system: the proton-coupled peptide transporter-1 (PepT1) encoded from the soluble carrier protein Slc15a1. In mammals, intestinal expression of PepT1/Slc15a1 oscillates during the daily feeding cycle. Although the oscillation in the intestinal expression of PepT1/Slc15a1 is suggested to be controlled by molecular components of circadian clock, we demonstrated here that bile acids regulated the oscillation of PepT1/Slc15a1 expression through modulating the activity of peroxisome proliferator-activated receptor α (PPARα). Nocturnally active mice mainly consumed their food during the dark phase. PPARα activated the intestinal expression of Slc15a1 mRNA during the light period, and protein levels of PepT1 peaked before the start of the dark phase. After food intake, bile acids accumulated in intestinal epithelial cells. Intestinal accumulated bile acids interfered with recruitment of co-transcriptional activator CREB-binding protein/p300 on the promoter region of Slc15a1 gene, thereby suppressing PPARα-mediated transactivation of Slc15a1. The timedependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the intestinal expression of PepT1/Slc15a1 during the daily feeding cycle that led to circadian changes in the intestinal absorption of small peptides. These findings suggest a molecular clock-independent mechanism by which bile acid-regulated PPARα activity governs the circadian expression of intestinal peptide transporter.

DOI： 10.1074/jbc.M114.577023

Language：English  

Parkinson's disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic neurons in the substantia nigra and dopamine depletion in the striatum. Although the motor symptoms are still regarded as the main problem, non-motor symptoms in PD also markedly impair the quality of life. Several non-motor symptoms, such as sleep disturbances and depression, are suggested to be implicated in the alteration in circadian clock function. In this study, we investigated circadian disruption and the mechanism in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. MPTP-treated mice exhibited altered 24-h rhythms in body temperature and locomotor activity. In addition, MPTP treatment also affected the circadian clock system at the genetic level. The exposure of human neuroblastoma cells (SH-SY5Y) to 1-metyl-4-phenylpyridinium (MPP⁺) increased or decreased the mRNA levels of several clock genes in a dose-dependent manner. MPP⁺-induced changes in clock genes expression were reversed by Compound C, an inhibitor of AMP-activated protein kinase (AMPK). Most importantly, addition of ATP to the drinking water of MPTP-treated mice attenuated neurodegeneration in dopaminergic neurons, suppressed AMPK activation and prevented circadian disruption. The present findings suggest that the activation of AMPK caused circadian dysfunction, and ATP may be a novel therapeutic strategy based on the molecular clock in PD.

DOI： 10.1007/s12017-012-8214-x

Language：English  

The dopamine D3 receptor (DRD3) in the ventral striatum is thought to influence motivation and motor functions. Although the expression of DRD3 in the ventral striatum has been shown to exhibit 24-hour variations, the mechanisms underlying the variation remain obscure. Here, we demonstrated that molecular components of the circadian clock act as regulators that control the 24-hour variation in the expression of DRD3. The transcription of DRD3 was enhanced by the retinoic acid-related orphan receptor a (RORa), and its activation was inhibited by the orphan receptor REV-ERBα, an endogenous antagonist of RORa. The serum or dexamethasone-induced oscillation in the expression of DRD3 in cells was abrogated by the downregulation or overexpression of REV-ERBα, suggesting that REV-ERBα functions as a regulator of DRD3 oscillations in the cellular autonomous clock. Chromatin immunoprecipitation assays of the DRD3 promoter indicated that the binding of the REVERBα protein to the DRD3 promoter increased in the early dark phase. DRD3 protein expression varied with higher levels during the dark phase. Moreover, the effects of the DRD3 agonist 7-hydroxy-N,N-dipropyl-2-aminotetralin (7-OHDPAT)- induced locomotor hypoactivity were significantly increased when DRD3 proteins were abundant. These results suggest that RORa and REV-ERBα consist of a reciprocating mechanism wherein RORa upregulates the expression of DRD3, whereas REV-ERBα periodically suppresses the expression at the time of day when REV-ERBα is abundant. Our present findings revealed that a molecular link between the circadian clock and the function of DRD3 in the ventral striatum acts as a modulator of the pharmacological actions of DRD3 agonists/antagonists.

DOI： 10.1124/mol.112.083535

Language：English  

The sensitivity of cancer cells to chemotherapeutic agents varies according to circadian time. Most chemotherapeutic agents ultimately cause cell death through cell-intrinsic pathways as an indirect consequence of DNA damage. The p53 tumor suppressor gene (TRP53) configures the cell deaths induced by chemotherapeutic agents. In this study, we show that the transcription factor ATF4, a component of the mammalian circadian clock, functions in circadian accumulation of p53 protein in tumor cells. In murine fibroblast tumor cells, ATF4 induced the circadian expression of p19ARF (Cdkn2a). Oscillation of p19ARF interacted in a time-dependent manner with MDM2, a specific ubiquitin ligase of p53, resulting in a rhythmic prevention of its degradation by MDM2. Consequently, the half-life of p53 protein varied in a circadian timedependent manner without variation in mRNA levels. The p53 protein accumulated during those times when the p19ARF-MDM2 interaction was facilitated. Notably, the ability of the p53 degradation inhibitor nutlin-3 to kill murine fibroblast tumor cells was enhanced when the drug was administered at those times of day during which p53 had accumulated. Taken together, these results suggested that ATF4-mediated regulation of the p19ARF-MDM2 pathway underlies the circadian accumulation of p53 protein in malignant cells. Furthermore, they suggest an explanation for how the sensitivity of cancer cells to chemotherapeutic agents is enhanced at those times of day when p53 protein has accumulated, as a result of circadian processes controlled by ATF4.

DOI： 10.1158/0008-5472.CAN-12-2492

Language：English  

In the animal model of brain metastasis using human lung squamous cell carcinoma-derived cells (HARA-B) inoculated into the left ventricle of the heart of nude mice, metastasized tumor cells and brain resident cells interact with each other. Among them, tumor cells and astrocytes have been reported to stimulate each other, releasing soluble factors from both sides, subsequently promoting tumor growth significantly. Among the receptors for soluble factors released from astrocytes, only IL-6 receptor (IL-6R) on tumor cells was up-regulated during the activation with astrocytes. Application of monoclonal antibody against human IL-6R (tocilizumab) to the activated HARA-B cells, the growth of HARA-B cells stimulated by the conditioned medium of HARA-B/astrocytes was significantly inhibited. Injecting tocilizumab to animal models of brain metastasis starting at three weeks of inoculation of HARA-B cells, two times a week for three weeks, significantly inhibited the size of the metastasized tumor foci. The up-regulated expression of IL-6R on metastasized lung tumor cells was also observed in the tissue from postmortem patients. These results suggest that IL-6R on metastasized lung tumor cells would be a therapeutic target to inhibit the growth of the metastasized lung tumor cells in the brain.

DOI： 10.3390/ijms14010515

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

Transferrin receptor 1(TfR1) is a key cell surface molecule that regulates the uptake of iron-bound transferrin. TfR1 expression is higher in tumor cells than in normal cells. Thus, intracellular targeting using iron-saturated Tf as a ligand for TfR-mediated endocytosis has attracted attention. TfR1 in colon cancer-bearing mice exhibits a 24-hour rhythm in mRNA and protein levels. The clock-controlled gene c-MYC rhythmically activate the transcription of the TfR1 gene. In addition, the cyclical accumulation of TfR1 causes dosing time-dependent changes in the intratumoral delivery of drug by receptor-mediated endocytosis. Identification of the circadian properties of molecules that are targeted by ligand-directed DDS may aid the choice of the most appropriate time of day for their administration.

Language：English  

Background: Overdosed acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) causes severe liver injury. We examined the effects of ozagrel, a selective thromboxane A₂ (TXA₂) synthase inhibitor, on liver injury induced by APAP overdose in mice.Methods: Hepatotoxicity was induced to ICR male mice by an intraperitoneal injection with APAP (330 mg/kg). The effects of ozagrel (200 mg/kg) treatment 30 min after the APAP injection were evaluated with mortality, serum alanine aminotransferase (ALT) levels and hepatic changes, including histopathology, DNA fragmentation, mRNA expression and total glutathione contents. The impact of ozagrel (0.001-1 mg/mL) on cytochrome P450 2E1 (CYP2E1) activity in mouse hepatic microsome was examined. RLC-16 cells, a rat hepatocytes cell line, were exposed to 0.25 mM N-acetyl-p-benzoquinone imine (NAPQI), a hepatotoxic metabolite of APAP. In this model, the cytoprotective effects of ozagrel (1-100 muM) were evaluated by the WST-1 cell viability assay.Results: Ozagel treatment significantly attenuated higher mortality, elevated serum alanine aminotransferase levels, excessive hepatic centrilobular necrosis, hemorrhaging and DNA fragmentation, as well as increase in plasma 2,3-dinor thromboxane B₂ levels induced by APAP injection. Ozagrel also inhibited the hepatic expression of cell death-related mRNAs induced by APAP, such as jun oncogene, FBJ osteosarcoma oncogene (fos) and C/EBP homologous protein (chop), but did not suppress B-cell lymphoma 2-like protein11 (bim) expression and hepatic total glutathione depletion. These results show ozagrel can inhibit not all hepatic changes but can reduce the hepatic necrosis. Ozagrel had little impact on CYP2E1 activity involving the NAPQI production. In addition, ozagrel significantly attenuated cell injury induced by NAPQI in RLC-16.Conclusions: We demonstrate that the TXA₂ synthase inhibitor, ozagrel, dramatically alleviates liver injury induced by APAP in mice, and suggest that it is a promising therapeutic candidate for the treatment of APAP-induced liver injury.

DOI： 10.1186/1471-230X-13-21

Language：English  

Objectives: The use of generic medicines is generally recommended for reducing medical costs. However, many doctors and pharmacists do not trust the quality, efficacy and safety of some generic medicines. Loxonin® containing loxoprofen sodium is widely used as an antipyretics analgesic, and Lobu® is the generic form of Loxonin® The pharmaceutical and therapeutic equivalence of Lobu® was compared to Loxonin® Methods: The dissolution rate of loxoprofen sodium from Lobu® was compared to that of Loxonin®. In addition, clinical data of the patients treated with Lobu® in Fukuoka Memorial Hospital was compared to those treated with Loxonin® in Fukuoka University Chikushi Hospital. The number of patients treated with Lobu® and Loxonin® was 76 and 79, and the mean age was 45.8 years old and 47.5 years old, respectively. Results: Lobu® did not meet the standard of the dissolution test of loxoprofen sodium at a pH of 1.2. However, the analgesic effect of Lobu® on patients undergone orthopedic surgery was equal to that of Loxonin®. In addition, severe hematopoietic injury, hepatic dysfunction, or renal dysfunction were not observed during Lobu® treatment. Conclusions: Although Lobu® is not 100&#37; pharmaceutically equivalent to Loxonin® the efficacy and safety are considered to be equivalent to Loxonin®.

Language：English  

Serotonin (5-HT) transporter (5-HTT) plays a key role in the control of 5-HT neuronal activity by reuptaking extracellular 5-HT from the synapse cleft. We have previously demonstrated that 5-HTT mRNA expression levels and its uptake activity in the mouse midbrain are significantly higher in the dark phase than those in the light phase. However, the molecular mechanisms of time-dependent expression of 5-HTT have not been clarified. In this study, expression of 5-HTT mRNA in the mouse midbrain showed a significant 24-h rhythm and was higher in the dark phase. Although such an oscillation was eliminated by a Clock gene mutation, CLOCK and BMAL1 did not activate 5-HTT transcription in the luciferase reporter assay. Activating transcription factor-4 (ATF4), a member of the ATF/cAMP response element (CRE)-binding protein family, is a component responsible for sustaining circadian oscillations of CRE-mediated gene expression. ATF4 significantly activated 5-HTT transcription in vitro and time dependently bound to the CRE site in the 5-HTT promoter in the mouse midbrain. In addition, mutation of the Clock gene disrupted temporal binding of ATF4 to the CRE site in the 5-HTT promoter. These results indicated that the circuit of circadian-basis molecular regulation between the clockwork system and mouse 5-HTT gene was connected by the ATF4 signaling pathway.

DOI： 10.1124/mol.112.079079

Language：English  

ABCG2, encoding breast cancer resistance protein (BCRP), is a member of the ATP-binding cassette transporter family and is often associated with cancer chemotherapeutic resistance. BCRP is also expressed in a variety of normal cells and acts as a xenobiotic efflux transporter. Because intestinal BCRP limits systemic exposure to xenobiotics, alterations in the function and expression of this transporter could account for part of the variation in oral drug absorption. In this study, we show that ATF4, a molecular component of the circadian clock, induces circadian expression of the Abcg2 gene in mouse small intestine. Three types of leader exons (termed exons 1A, 1B, and 1C) are identified in the 5′-untranslated region of mouse Abcg2 transcripts. The exon 1B-containing Abcg2 transcript was the only isoform detected in mouse small intestine, and its mRNA levels oscillated in a circadian time-dependent manner. ATF4 bound time-dependently to the cAMP response element within the exon 1B promoter region of the Abcg2 gene, thereby causing the oscillation of BCRP protein abundance and its efflux pump function. The circadian clock-ATF4 pathway appears to enhance the function of BCRP during a specific time window and to modulate intestinal drug absorption. Our findings suggest a mechanism underlying circadian change in xenobiotic detoxification.

DOI： 10.1074/jbc.M111.333377

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

Differentiated embryo chondrocyte-2 (DEC2), also known as bHLHE41 or Sharp1, is a pleiotropic transcription repressor that controls the expression of genes involved in cellular differentiation, hypoxia responses, apoptosis, and circadian rhythm regulation. Although a previous study demonstrated that DEC2 participates in the circadian control of hepatic metabolism by regulating the expression of cytochrome P450, the molecular mechanism is not fully understood. We reported previously that brief exposure of HepG2 cells to 50% serum resulted in 24-h oscillation in the expression of CYP3A4 as well as circadian clock genes. In this study, we found that the expression of CYP2D6, a major drug-metabolizing enzyme in humans, also exhibited a significant oscillation in serum-shocked HepG2 cells. DEC2 interacted with CCAAT/enhancer-binding protein (C/EBPα), accompanied by formation of a complex with histone deacetylase-1, which suppressed the transcriptional activity of C/EBPα to induce the expression of CYP2D6. The oscillation in the protein levels of DEC2 in serum-shocked HepG2 cells was nearly antiphase to that in the mRNA levels of CYP2D6. Transfection of cells with small interfering RNA against DEC2 decreased the amplitude of CYP2D6 mRNA oscillation in serum-shocked cells. These results suggest that DEC2 periodically represses the promoter activity of CYP2D6, resulting in its circadian expression in serum-shocked cells. DEC2 seems to constitute a molecular link through which output components from the circadian clock are associated with the time-dependent expression of hepatic drug-metabolizing enzyme.

DOI： 10.1124/mol.111.076406

Language：English  

Many cancers overexpress ATF4, a stress-induced transcription factor that promotes cell survival under hypoxic conditions and other stresses of the tumor microenvironment, but the potential contributions of ATF4 to oncogenesis itself have been little explored. Here, we report that ATF4 promotes oncogene-induced neoplastic transformation by suppressing the expression of cellular senescence-associated genes. Strikingly, primary embryo fibroblasts from ATF4-deficient mice were resistant to transformation by coexpression of H-ras^V12 and SV40 large T antigen. In wild-type cells these oncogenes induced expression of the murine Atf4 gene along with the cyclin-dependent kinase inhibitor Cdkn2a, which encodes the cell senescence-associated proteins p16INK4 and p19ARF. Elevated levels of ATF4 were sufficient to suppress expression of these proteins and drive oncogenic transformation. Conversely, genetic ablation of ATF4 led to constitutive expression of p16INK4a and p19ARF, triggering cellular senescence. Our findings define a central function for ATF4 in promoting oncogenic transformation by suppressing a central pathway of cellular senescence.

DOI： 10.1158/0008-5472.CAN-11-1891

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

Objectives: The use of generic medicines is generally recommended for reducing medical costs. However, many doctors and pharmacists do not trust the quality, efficacy and safety of some generic medicines. Loxonin® containing loxoprofen sodium is widely used as an antipyretics analgesic, and Lobu® is the generic form of Loxonin® The pharmaceutical and therapeutic equivalence of Lobu® was compared to Loxonin® Methods: The dissolution rate of loxoprofen sodium from Lobu® was compared to that of Loxonin®. In addition, clinical data of the patients treated with Lobu® in Fukuoka Memorial Hospital was compared to those treated with Loxonin® in Fukuoka University Chikushi Hospital. The number of patients treated with Lobu® and Loxonin® was 76 and 79, and the mean age was 45.8 years old and 47.5 years old, respectively. Results: Lobu® did not meet the standard of the dissolution test of loxoprofen sodium at a pH of 1.2. However, the analgesic effect of Lobu® on patients undergone orthopedic surgery was equal to that of Loxonin®. In addition, severe hematopoietic injury, hepatic dysfunction, or renal dysfunction were not observed during Lobu® treatment. Conclusions: Although Lobu® is not 100% pharmaceutically equivalent to Loxonin® the efficacy and safety are considered to be equivalent to Loxonin®.

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

The expression of genes involved in xenobiotic detoxification is under the control of the circadian clock. The aryl hydrocarbon receptor (AhR) is one of the transcription factors responsible for the induction of detoxification enzymes in response to xenobiotic toxins, and the expression of AhR has been suggested to be regulated by a circadian oscillator. In this study, we investigated whether toxin-mediated activation of the AhR signaling pathway was modulated by CLOCK protein, a key component of the mammalian circadian clock. The expression of AhR and its DNA binding ability in the lungs of wild-type mice showed significant 24-h oscillation. . Clock mutant (. Clk/Clk) mice, producing CLOCK protein deficient in transcriptional activity, failed to show significant oscillation in the expression of AhR. The mRNA levels of . AhR in the lungs of . Clk/Clk mice were significantly lower than in wild-type mice. A single intraperitoneal injection of benzo[α]pyrene, a ligand of AhR, induced the expression of . Cyp1a1 in the lungs of wild-type mice, but the induction varied depending on the benzo[α]pyrene injection time. The dosing time-dependency of benzo[α]pyrene-induced . Cyp1a1 expression was also modulated by . Clock gene mutation. These findings suggest that CLOCK protein affects the toxin-induced expression of detoxification enzymes through modulating the activity of AhR. Our present findings provide a molecular link between the circadian clock and xenobiotic detoxification.

DOI： 10.1016/j.tox.2011.09.007

Language：English  

Many pathophysiological circumstances vary during 24h periods. Many physiologic processes undergo biological rhythms, including the sleep-wake rhythm and metabolism. Disruptive effect in the 24h variations can manifest as the emergence or exacerbation of pathological conditions. So, chronotherapeutics is gaining increasing interest in experimental biology, medicine, pharmacy, and drug delivery. This science and the plethora of information should be used intelligently for optimizing the effectiveness and safety of the drug, relying on the timing of drug intake. These chronopharmacological findings are affected by not only the pharmacodynamics but also pharmacokinetics of drugs. The mammalian circadian pacemaker is located in the suprachiasmatic nucleus. The molecular mechanisms are associated with Clock genes that control the circadian rhythms in physiology, pathology, and behavior. Clock controls several diseases such as metabolic syndrome, cancer, and so on. CLOCK mutation influences the expression of both rhythmic and nonrhythmic genes in wild-type tissues. These genotypic changes lead to phenotypic changes, affecting the drug pharmacokinetic and pharmacodynamic parameters. This review is intended to elaborate system regulating biological rhythms and the applicability in pharmaceutics from viewpoints of the intraindividual and interindividual variabilities of Clock genes.

DOI： 10.1002/jps.22656

Language：English  

Activating transcription factor (ATF)/cAMP-response element (CRE)-binding (CREB) proteins induce the CRE-mediated gene transcription depending on the cAMP stimulation. cAMP-dependent signaling oscillates in a circadian manner, which in turn also sustains core oscillation machinery of the circadian clock. Here, we show that among the ATF/CREB family proteins, ATF4 is essential for the circadian expression of the Period2 (Per2) gene, a key component of the circadian clock. Transcription of the Atf4 gene was regulated by core components of the circadian clock, and its expression exhibited circadian oscillation in mouse tissues as well as embryonic fibroblasts. ATF4 bound to the CRE of the Per2 promoter in a circadian time-dependent manner and periodically activated the transcription of the Per2 gene. Consequently, the oscillation of the Per2 expression was attenuated in embryonic cells prepared from Atf4-null mice. Furthermore, the loss of ATF4 also disrupted the rhythms in the expression of other clock genes. These results suggest that ATF4 is a component responsible for sustaining circadian oscillation of CRE-mediated gene expression and also constitute a molecular link connecting cAMP-dependent signaling to the circadian clock.

DOI： 10.1074/jbc.M111.258970

Language：English  

The Clock gene is a core clock factor that plays an essential role in generating circadian rhythms. In the present study, it was investigated whether the Clock gene affects the response to diethylnitrosamine (DEN)-induced cytotoxicity using mouse primary hepatocytes. DEN-induced cytotoxicity, after 24. h exposure, was caused by apoptosis in hepatocytes isolated from wild-type mouse. On the other hand, Clock mutant mouse (Clk/. Clk) hepatocytes showed resistance to apoptosis. Because apoptosis is an important pathway for suppressing carcinogenesis after genomic DNA damage, the mechanisms that underlie resistance to DEN-induced apoptosis were examined in Clk/. Clk mouse hepatocytes. The mRNA levels of metabolic enzymes bioactivating DEN and apoptosis-inducing factors before DEN exposure were lower in Clk/. Clk cells than in wild-type cells. The accumulation of p53 and Ser15 phosphorylated p53 after 8. h DEN exposure was seen in wild-type cells but not in Clk/. Clk cells. Caspase-3/7 activity was elevated during 24. h DEN exposure in wild-type cells but not in Clk/. Clk cells. In addition, resistance to DEN-induced apoptosis in Clk/. Clk cells affected the cell viability. These studies suggested that the lower expression levels of metabolic enzymes bioactivating DEN and apoptosis inducing factors affected the resistance to DEN-induced apoptosis in Clk/. Clk cells, and the Clock gene plays an important role in cytotoxicity induced by DEN.

DOI： 10.1016/j.tox.2010.12.005

Language：English  

DOI： 10.1254/fpj.137.115

Language：English  

Objectives: The use of generic drugs is recommended in order to reduce medical costs. However, many doctors and pharmacists do not trust the quality, efficacy and safety of some generic drugs. Bezator® SR containing bezafibrate is widely used for the treatment of hyperlipidemia, and besastar® SR is the generic drug of bezator® SR. We herein compared the pharmaceutical and therapeutic equivalence of besastar® SR with those of bezator® SR. Methods: The elution rate of bezafibrate from besastar® SR was compared to that of bezator® SR. In addition, the clinical data of the patients treated with besastar® SR at Fukuoka Memorial Hospital were compared with those treated with bezator® SR at Fukuoka University Chikushi Hospital. Results: One of six lots of besastar® SR did not satisfy the standards of elution of bezafibrate. However, the effect of besastar® SR on the blood triglyceride concentration after 4-12 weeks was equivalent to that of bezator® SR. In addition, in a manner similar to that of bezator® SR, besastar® SR did not induce hematopoietic injury, hepatic dysfunction, renal dysfunction or rhabdomyolysis during the period from 4-12 weeks after the initiation of treatment. Conclusions: Although besastar® SR is not 100&#37; pharmaceutically equivalent to bezator® SR, the efficacy and safety of this generic drug are considered to be equivalent to those of bezator® SR.

Language：English  

Many pathophysiological circumstances vary during 24h periods. Many physiologic processes undergo biological rhythms, including the sleep-wake rhythm and metabolism. Disruptive effect in the 24h variations can manifest as the emergence or exacerbation of pathological conditions. So, chronotherapeutics is gaining increasing interest in experimental biology, medicine, pharmacy, and drug delivery. This science and the plethora of information should be used intelligently for optimizing the effectiveness and safety of the drug, relying on the timing of drug intake. These chronopharmacological findings are affected by not only the pharmacodynamics but also pharmacokinetics of drugs. The mammalian circadian pacemaker is located in the suprachiasmatic nucleus. The molecular mechanisms are associated with Clock genes that control the circadian rhythms in physiology, pathology, and behavior. Clock controls several diseases such as metabolic syndrome, cancer, and so on. CLOCK mutation influences the expression of both rhythmic and nonrhythmic genes in wild-type tissues. These genotypic changes lead to phenotypic changes, affecting the drug pharmacokinetic and pharmacodynamic parameters. This review is intended to elaborate system regulating biological rhythms and the applicability in pharmaceutics from viewpoints of the intraindividual and interindividual variabilities of Clock genes.

DOI： 10.1002/jps.22656

Language：English  

Acute thrombotic events frequently occur in the early morning among hyperlipidemic patients. The activity of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of the fibrinolytic system, oscillates daily, and this is considered one mechanism that underlies the morning onset of acute thrombotic events in hyperlipidemia. Although several studies have reported the expression of the PAI-1 gene is under the control of the circadian clock system, the molecular mechanism of the circadian transactivation of PAI-1 gene under hyperlipidemic conditions remains to be elucidated. Here, the authors investigated whether hyperlipidemia induced by a high-fat diet (HFD) enhances the daily oscillation of plasma PAI-1 activity in mice. The mRNA levels of the PAI-1 gene were increased and rhythmically fluctuated with high-oscillation amplitude in the livers of wild-type mice fed with the HFD. Circadian expression of proxisome proliferator-activated receptor-α (PPARα) mRNA was also augmented as well as that of PAI-1. Chromatin immunoprecipitaion showed the HFD-induced hyperlipidemia significantly increased the binding of PPARα to the PAI-1 promoter. Luciferase reporter analysis using primary hepatocytes revealed CLOCKBMAL1-mediated PAI-1 promoter activity was synergistically enhanced by cotransfection with PPARαretinoid X receptor-α (RXRα), and this synergistic transactivation was repressed by negative limbs of the circadian clock, PERIOD2 and CRYPTOCHROME1. As expected, HFD-induced PAI-1 mRNA expression was significantly attenuated in PPARα-null mice. These results suggest a molecular link between the circadian clock and lipid metabolism system in the regulation of PAI-1 gene expression, and provide an aid for understanding why hyperlipidemia increases the risk of acute thrombotic events in the morning.

DOI： 10.3109/07420528.2010.515324

Language：English  

Objectives: The use of generic drugs is recommended for reducing of medical costs. However, many doctors and pharmacists do not trust the quality, efficacy and safety of some generic drugs. Glimicron® containing gliclazide is widely used for the treatment of diabetes mellitus, and glimiran® is the generic drug of glimicron®. Then, the pharmaceutical and therapeutic equivalence of glimiran® was compared to glimicron®. Methods: The elution rate of gliclazide from glimiran® was compared to that of glimicron®. In addition, clinical data of the patients treated with glimiran® in Fukuoka Memorial Hospital was compared to those treated with glimicron® in Fukuoka University Chikushi Hospital. The number of patients treated with glimiran® and glimicron® was 28 and 56, and the mean age was 63.9 years old and 60.3 years old, respectively. Results: One of two lots of glimiran® did not satisfy the standards of elution of gliclazide. However, the effect of glimiran® on both blood sugar and hemoglobin A1c after 8-12 weeks was equivalent to glimicron®. In addition, in a manner similar to that of glimicron®, glimiran® did not induce hematopoietic injury, hepatic dysfunction or renal dysfunction during the period from 8-12 weeks after the initiation of treatment. Conclusions: Although glimiran® is not 100% pharmaceutically equivalent to glimicron®, the efficacy and safety are considered to be equivalent to glimicron®.

Language：English  

Background: Neuropathic pain is characterized by hypersensitivity to innocuous stimuli (tactile allodynia) that is nearly always resistant to NSAIDs or even opioids. Gabapentin, a GABA analogue, was originally developed to treat epilepsy. Accumulating clinical evidence supports the effectiveness of this drug for diverse neuropathic pain. In this study, we showed that the anti-allodynic effect of gabapentin was changed by the circadian oscillation in the expression of its target molecule, the calcium channel α2δ-1 subunit.Results: Mice were underwent partial sciatic nerve ligation (PSL) to create a model of neuropathic pain. The paw withdrawal threshold (PWT) in PSL mice significantly decreased and fluctuated with a period length about 24 h. The PWT in PSL mice was dose-dependently increased by intraperitoneal injection of gabapentin, but the anti-allodynic effects varied according to its dosing time. The protein levels of α2δ-1 subunit were up-regulated in the DRG of PSL mice, but the protein levels oscillated in a circadian time-dependent manner. The time-dependent oscillation of α2δ-1 subunit protein correlated with fluctuations in the maximal binding capacity of gabapentin. The anti-allodynic effect of gabapentin was attenuated at the times of the day when α2δ-1 subunit protein was abundant.Conclusions: These findings suggest that the dosing time-dependent difference in the anti-allodynic effects of gabapentin is attributable to the circadian oscillation of α2δ-1 subunit expression in the DRG and indicate that the optimizing its dosing schedule helps to achieve rational pharmacotherapy for neuropathic pain.

DOI： 10.1186/1744-8069-6-83

Language：English  

Hypoxia-induced gene expression frequently occurs in malignant solid tumors because they often have hypoxic areas in which circulation is compromised due to structurally disorganized blood vessels. Hypoxia-response elements (HREs) are responsible for activating gene transcription in response to hypoxia. In this study, we constructed a hypoxia-response plasmid vector producing short hairpin RNA (shRNA) against B-cell leukemia/lymphoma-2 (bcl-2), an anti-apoptotic factor. The hypoxia-response promoter was made by inserting tandem repeats of HREs upstream of cytomegalovirus (CMV) promoter (HRE-CMV). HRE-CMV shbcl-2 vector consisted of bcl-2 shRNA under the control of HRE-CMV promoter. In hypoxic mouse rectum carcinoma cells (colon-26), the production of bcl-2 shRNA driven by HRE-CMV promoter was approximately 2-fold greater than that driven by CMV promoter. A single intratumoral (i.t.) injection of 40 μ g HRE-CMV shbcl-2 to colon-26 tumor-bearing mice caused apoptotic cell death, and repetitive treatment with HRE-CMV shbcl-2 (40 μ g/mouse, i.t.) also significantly suppressed the growth of colon-26 tumor cells implanted in mice. Apoptotic and anti-tumor effects were not observed in tumor-bearing mice treated with CMV shbcl-2. These results reveal the ability of HRE-CMV shbcl-2 vector to suppress the expression of bcl-2 in hypoxic tumor cells and suggest the usefulness of our constructed hypoxia-response plasmid vector to treat malignant tumors.

DOI： 10.1254/jphs.10054FP

Language：English  

The abundance of cell surface levels of transferrin receptor 1 (TfR1), which regulates the uptake of iron-bound transferring, correlates with the rate of cell proliferation. Because TfR1 expression is higher in cancer cells than in normal cells, it offers a target for cancer therapy. In this study, we found that the expression of TfR1 in mouse colon cancer cells was affected by the circadian organization of the molecular clock. The core circadian oscillator is composed of an autoregulatory transcription-translation feedback loop, in which CLOCK and BMAL1 are positive regulators and the Period (Per), Cryptochrome (Cry), and Dec genes act as negative regulators. TfR1 in colon cancer-bearing mice exhibited a 24-hour rhythm in mRNA and protein levels. Luciferase reporter analysis and chromatin immunoprecipitation experiments suggested that the clock-controlled gene c-MYC rhythmically activated the transcription of the TfR1 gene. Platinum incorporation into tumor DNA and the antitumor efficacy of transferrin-conjugated liposome-delivered oxaliplatin could be enhanced by drug administration at times when TfR1 expression increased. Our findings suggest that the 24-hour rhythm of TfR1 expression may form an important aspect of strategies for TfR1-targeted cancer therapy.

DOI： 10.1158/0008-5472.CAN-10-0184

Language：English  

A functional interaction between peroxisome proliferator-activated receptor α (PPARα) and components of the circadian clock has been suggested, but whether these transcriptional factors interact to regulate the expression of their target genes remains obscure. Here we used a PPARα ligand, bezafibrate, to search for PPARα-regulated genes that are expressed in a CLOCK-dependent circadian manner. Microarray analyses using hepatic RNA isolated from bezafibrate treated-wild type, Clock mutant (Clk/Clk), and PPARα-null mice revealed that 136 genes are transcriptionally regulated by PPARα in a CLOCK-dependent manner. Among them, we focused on the plasminogen activator inhibitor-1 (PAI-1) gene, because its expression typically shows circadian variation, and it has transcriptional response elements for both PPAR and CLOCK. The bezafibrate-induced expression of PAI-1 mRNA was attenuated in Clk/Clk mice and in PPARα-null mice. The protein levels of PPARα were reduced in Clk/Clk hepatocytes. However, the overexpression of PPARα could not rescue bezafibrate-induced PAI-1 expression in Clk/Clk hepatocytes, suggesting that impaired bezafibrate-induced PAI-1 expression in Clk/Clk mice is not due to reduced PPARα expression. Luciferase reporter and chromatin immunoprecipitation analyses using primary hepatocytes demonstrated that DNA binding of both PPARα and CLOCK is essential for bezafibrate-induced PAI-1 gene expression. Pull-down assays in vitro showed that both PPARαand its heterodimerized partner retinoic acid receptor-α can serve as potential interaction targets of CLOCK. The present findings revealed that molecular interaction between the circadian clock and the lipid metabolism regulator affects the bezafibrate-induced gene expression.

DOI： 10.1124/mol.110.064402

Language：English  

In all living organisms, one of the most indispensable biological functions is the circadian clock (suprachiasmatic nuclei; SCN), which acts like a multifunction timer to regulate homeostatic systems such as sleep and activity, hormone levels, appetite, and other bodily functions with 24. h cycles. Circadian rhythms regulate diverse physiologic processes, including homeostatic functions of steroid hormones and their receptors. Perturbations of these rhythms are associated with pathogenic conditions such as depression, diabetes and cancer. Clock genes are identified as the genes that ultimately control a vast array of circadian rhythms in physiology and behavior. Clock gene regulates several diseases such as cancer, metabolic syndrome and sleep etc. CLOCK mutation affects the expression of rhythmic genes in wild-type (WT) tissue, but also affects that of non-rhythmic genes. On the other hand, the change of the drug pharmacodynamic and pharmacokinetic (PK/PD) parameters are influenced by not only inter-individual variability but also intra-individual variabilities of medications. Identification of a rhythmic marker for selecting dosing time will lead to improved progress and diffusion of chronopharmacotherapy. The mechanisms underlying chronopharmacological findings should be clarified from viewpoint of clock genes. On the other hand, several drugs have an effect on molecular clock. Thus, the knowledge of intra- and inter-individual variability of molecular clock should be applied for the clinical practice. Therefore, we introduce the regulatory system of biological rhythm from viewpoints of clock genes and the possibility of pharmacotherapy based on the intra- and inter-individual variability of clock genes.

DOI： 10.1016/j.addr.2010.04.005

Language：English  

O⁶-methylguanine-DNA methyltransferase (MGMT) plays a crucial role in the defense against the alkylating agent-induced cytotoxic lesion O ⁶-alkylguanine in DNA. Although a significant circadian variation in MGMT activity has been found in the liver of mice, the exact mechanism of the variation remains poorly understood. In this study, we present evidence that glucocorticoids were required for the 24-h oscillation of MGMT expression in mouse liver. The exposure of mouse hepatic cells (Hepa1-6) to dexamethasone (DEX) significantly increased the mRNA levels of MGMT in a dose-dependent manner. The DEX-induced increase in MGMT expression was reversed by concomitant treatment with RU486 [11β-[p-(dimethylamino) phenyl]-17β-hydroxy-17- (1-propynyl)estra-4,9-dien-3-one], a glucocorticoid receptor antagonist. The mRNA levels of MGMT and its enzymatic activity in the liver of mice showed significant 24-h oscillations, which were not observed in adrenalectomized mice. A single administration of DEX to adrenalectomized mice significantly increased the mRNA levels of MGMT in the liver. These findings suggest that the 24-h oscillation in the hepatic expression of MGMT is caused by the endogenous rhythm of glucocorticoid secretion. Dacarbazine (DTIC), a potent O⁶- guanine-alkylating agent, causes serious hepatotoxicity accompanied by hepatocellular necrosis and hepatic vein thrombosis. DTIC-induced hepatotoxicity in mice was attenuated by administering the drug at the time of day when MGMT expression was abundant. The present findings suggest that glucocorticoid- regulated oscillation in the hepatic MGMT expression is the underlying cause of dosing time-dependent changes in DTIC-induced hepatotoxicity.

DOI： 10.1124/jpet.110.165597

Language：English  

The mammalian circadian pacemaker stays in the paired suprachiasmatic nuclei (SCN). Recent several studies reveal that the circadian rhythms of physiology and behavior are controlled by clock genes. In addition, the effectiveness and toxicity of many drugs vary depending on dosing time associated with 24-h rhythms of biochemical, physiological, and behavioral processes under the control of the circadian clock. Acetaminophen (APAP) is a widely used analgesic drug, and is mainly biotransformed and eliminated as nontoxic conjugates with glucuronic acid and sulfuric acid. Only a small portion of the dose is mainly bioactivated by CYP2E1 to N-acetyl-p-benzoquinone imine (NAPQI), a reactive toxic intermediate. For APAP overdose, glucuronidation and sulfation are saturated and the formation of NAPQI increases. However, the exact mechanisms underlying the chronotoxicity of APAP have not been clarified yet. In the present study, we have clarified that there was a significant dosing time-dependent difference in hepatotoxicity induced by APAP in mice. The mechanism may be related to the rhythmicity of CYP2E1 activity and GSH conjugation. In additon, we investigated whether the liver transcription factor hepatic nuclear factor-1α (HNF-1α) and clock genes undergoing astriking 24-h rhythm in mouse liver contribute to the 24-h regulation of CYP2E1 activity. A significant 24-h rhythmicity was demonstrated for CYP2E1 activity, protein levels and mRNA levels. HNF-1α and clock genes may contribute to produce the 24-h rhythm of CYP2E1 mRNA levels. Metabolism by CYP and GSH conjugation are common metabolic pathways for many drugs such as APAP. These findings support the concept that choosing the most appropriate time of day to administer the drugs associated with metabolic rhythmicity such as CYP and GSH conjugation may reduce hepatotoxicity in experimental and clinical situations. 24-h rhythm of CYP2E1 activity was controlled by HNF-1α and clock gene, in a transcriptional level. Identification of rhythmic marker for selecting dosing time will lead improved progress and diffusion of chronopharmacotherapy.

DOI： 10.1248/yakushi.129.1357

Language：English  

Although Clock gene product, a component of the circadian pacemaker, has been suggested to participate in the regulation of cellular sensitivity to genotoxic stress, the underlying mechanism remains to be fully understood. In this study, we showed that Clock gene mutation modulates the sensitivity of hepatocytes to alkylating agent-induced genotoxic stress through altering the expression of N-methylpurine DNA glycosylase (MPG), the first enzyme in the base excision repair pathway. Neither wild-type nor Clock mutant (Clock/Clock) mice showed a significant 24-h variation in the hepatic expression of MPG. However, the mRNA and protein levels of MPG in the liver of Clock/Clock mice were significantly lower than those in wild-type liver. The cytotoxic effect of methyl methanesulfonate (MMS), a methylating agent, on primary cultured hepatocytes prepared from Clock/Clock mice was more potent than on wild-type hepatocytes, while overexpression of MPG in Clock/Clock hepatocytes restored their MMS sensitivity to the wild-type level. These findings suggest that the product of the Clock gene controls the sensitivity of cells to genotoxic stress through regulating the expression of the MPG gene. Our present findings would provide a molecular link between the circadian clock and DNA repair pathway.

DOI： 10.1016/j.bcp.2009.06.013

Language：English  

In clinical practice, glucocorticoids are often used with the aim of modulating the efficacy and toxicity of chemotherapeutic agents. However, how glucocorticoids modulate the pharmacological action of chemotherapeutic agents remains to be clarified. In this study, we generated glucocorticoid receptor (GR)-deficient rat-1 cells to investigate the role of GR in the regulation of cellular sensitivity to irinotecan hydrochloride (CPT-11). Treatment of wild-type rat-1 cells with dexamethasone (DEX) significantly enhanced the cytotoxic effect of CPT-11, whereas the treatment had little effect on the cytotoxicity of CPT-11 in GR-deficient cells. Topoisomerase-I activity in wild-type cells after concomitant treatment with DEX and CPT-11 was significantly lower than that after treatment with CPT-11 alone. DEX treatment also enhanced the inhibitory action of CPT-11 on the phosphatidylinositol 3-kinase-Akt signaling pathway in wild-type cells, accompanied by facilitating caspase-3 activity. These modulatory effects of DEX on the CPT-11-induced cytotoxicity were not observed in GR-deficient cells. Our present findings reveal the underlying mechanism by which GCs enhance the chemotherapeutic effect of CPT-11 and indicate the possibility that the dosage of CPT-11 could be reduced by the combination treatment with GCs, which may attenuate the adverse effect without decreasing anti-tumor activity. [Supplementary Figures: available only at http://dx.doi.org /10.1254/jphs.08219FP]

DOI： 10.1254/jphs.08219FP

Language：English  

Altered homeostatic regulation, including the disturbance of circadian rhythms, is often observed in patients undergoing interferon (IFN) therapy. We reported previously that IFN-α has the ability to modulate the circadian clock function at the molecular level and that the alteration of clock function could be overcome by changing the dosing schedule. In this study, we investigated the influence of IFN-α on the intrinsic biological rhythms in mice by comparing two dosing schedules, continuous administration and repetitive injection. Continuous administration of IFN-α to mice decreased the rhythm amplitude of locomotor activity, body temperature, leukocyte counts, and plasma corticosterone levels. The treatment also suppressed the oscillation in the expression of clock genes in the liver. On the other hand, modulation effects were scarcely observed in mice treated with repetitive injection of IFN-α. These results indicate that treatment with IFN-α does not always modulate the circadian clock function. This notion was also supported by in vitro findings that the inhibitory action of IFN-α on the expression of clock genes was dependent on its exposure time to cells. The alteration of clock function induced by IFN-α could be avoided by optimizing the dosing schedule.

DOI： 10.1016/j.lfs.2008.08.005

Language：English  

Circadian synchronization of cell proliferation is observed not only in normal healthy tissues but also in malignant solid tumors. However, the proliferation rhythm of tumor cells is often different from that of normal cells. We reported here that the peculiar rhythm of tumor cell proliferation was modulated by inhibition of platelet-derived growth factor (PDGF) signaling. DNA synthesis in tumor cells implanted in mice showed a 24-h oscillation apparently differing from that of normal bone marrow cells. Continuous administration of AG1295 (10 μg/h, s.c.), a PDGF receptor tyrosine kinase inhibitor, substantially suppressed DNA synthesis in the implanted tumor cells but not in the healthy bone marrow cells. During the administration of this drug, the rhythm of DNA synthesis in the tumor cells was synchronized with that in bone marrow cells. The present results suggest that the circadian rhythm of DNA synthesis in tumor cells is modulated by PDGF receptor signaling, which is activated following tumor progression. Because the rhythmic patterns of clock gene expression in tumor cells did not differ significantly from those in other healthy tissues, the enhanced signal transduction of PDGF receptor may cause an alteration in the rhythmicity of tumor cell proliferation without changing in the intracellular molecular clockwork.

DOI： 10.1254/jphs.08080FP

Language：English  

Cytochrome P450 2E1 (CYP2E1) is clinically and toxicologically important and exhibits 24-hour periodicity in its activity. In the present study, we investigated whether hepatic nuclear factor-1α (HNF-1α) and clock genes with a striking 24-hour rhythm in mouse liver contributed to the 24-hour regulation of CYP2E1 expression. The results demonstrated that the expression of CYP2E1 messenger RNA (mRNA) in the liver was affected by HNF-1α and the circadian organization of molecular clocks. The mRNA levels of CYP2E1 in the liver increased from the late light phase to the early dark phase. Luciferase reporter gene analysis revealed that HNF-1α activated CYP2E1 promoter activity, which was restricted by CRY1, a member of the circadian organization of molecular clocks. Repressor activity of CRY1 was observed on the HNF-1α binding site of the CYP2E1 promoter region with mutated E-box. Serum shock induced approximately 24-hour oscillation in CYP2E1 mRNA in HepG2. Transfection of HNF-1α and CRY1 small interfering RNA dampened the oscillation of CYP2E1 mRNA in HepG2. Chromatin immunoprecipitation assay in the CYP2E1 promoter indicated that HNF-1α binding to the CYP2E1 promoter increased from the late light phase to the early dark phase. Using the chromatin immunoprecipitation reimmunoprecipitation assay, time-dependent differences were demonstrated for CRY1 protein interaction with HNF-1α transcriptional complexes, including coactivator p300 on the HNF-1α binding site in the CYP2E1 promoter. Conclusion: Our results suggest that the transcription activator of HNF-1α acts periodically and the negative limbs of molecular clocks periodically inhibit CYP2E1 transcription, resulting in the 24-hour rhythm of its mRNA expression.

DOI： 10.1002/hep.22304

Language：English  

The circadian clock system is necessary to adapt endogenous physiological functions to daily variations in environmental conditions. Abnormality in circadian rhythms, such as the sleep-wake cycle and the timing of hormonal secretions, is implicated in various physiological and psychiatrical disorders. Recent molecular studies have revealed that oscillation in the transcription of specific clock genes plays a central role in the generation of 24 h cycles of physiology and behavior. It has been noticed that patients receiving chemotherapeutic agents experience disturbances in their behavioral and physical performances, including circadian rhythms. To explore the underlying mechanism of chemotherapeutic agent-induced disturbance of these rhythms, we investigated the influence of 5-fluorouracil (5-FU), one of the most widely used chemotherapeutic agents for the treatment of cancers, on the expression of clock genes. Treatment of cultured NIH3T3 cells with 5-FU for 48 h resulted in a significant reduction of mRNA levels of Period1 (Per1) and Period2 (Per2) without affecting cell viability; however, treatment with the same amount of uracil, a structural analog of 5-FU, had little effect on the expression of clock genes. Consistent with its inhibitory actions, continuous administration of 5-FU (2 mg/kg/h) to mice attenuated the oscillation in the expressions of Per1 and Per2 in the liver and suprachiasmatic nuclei, the center of the mammalian circadian clock. These results reveal a possible pharmacological action by the chemotherapeutic agent 5-FU on the circadian clock mechanism, which is the underlying cause of its adverse effects on 24-h rhythms of physiology and behavior.

DOI： 10.1016/j.bcp.2008.01.011

Language：English  

Hepatocyte growth factor (HGF) ameliorates liver injuries in hepatectomized cholestatic rats. On the other hand, the protein level of organic anion-transporting polypeptide (Oatp1), which is responsible for the uptake of bile salts into hepatocytes, decreases in cholestatic humans and rats. However, the relationship between the ameliorative effects of HGF and the decrease in Oatp1 levels in cholestasis remains to be understood. Therefore, in order to investigate this relationship, we evaluated the effects of HGF on the function and protein level of Oatp1. HGF treatment significantly increased the uptake of radiolabeled estradiol 17β-d-glucuronide ([³H]E₂17βG), a predominant Oatp1 substrate, in primary cultured rat hepatocytes. Additionally, there was an increase in the Oatp1 protein levels. The increased [³H]E₂17βG uptake was significantly inhibited by simultaneous incubation with the HGF receptor antibody and treatment with non-radiolabeled E₂17βG. However, inhibition by taurocholic acid, a Na⁺-taurocholate co-transporting polypeptide (Ntcp) substrate, was weaker than that caused by non-radiolabeled E₂17βG. Further, the increase was not altered by replacing Na⁺ in the medium with Li⁺. In the inhibition study, the increased [³H]E₂17βG uptake was inhibited by Oatp1 substrates, including bromosulfophthalein, ochratoxin A, and ouabain, but not by digoxin, which is an Oatp2-specific substrate. Furthermore, HGF did not alter the Oatp1 mRNA expression. In contrast, HGF treatment suppressed the ubiquitination of Oatp1 protein. In conclusion, this is the first report suggesting that HGF regulates Oatp1 protein level and that the ameliorative effects of HGF in cholestasis was induced, at least in part, by correcting the down-regulation of the Oatp1 protein level.

DOI： 10.1016/j.ejphar.2007.10.041

Language：English  

Background & Aims: P-glycoprotein, the product of the multidrug resistance (mdr) gene, functions as a xenobiotic transporter contributing to the intestinal barrier. Although intestinal expression of the mdr1a gene and its efflux pump function has been shown to exhibit 24-hour variation, the mechanism of the variations remains poorly understood. Here, we demonstrated that the molecular components of the circadian clock act as regulators to control 24-hour variation in the expression of the mdr1a gene. Methods: Luciferase reporter assay and gel mobility shift assay were used to study the mechanism of transcriptional regulation of the mdr1a gene by clock gene products. The messenger RNA levels and protein abundances in colon 26 cells and mouse intestine were measured by quantitative real-time polymerase chain reaction and Western blotting, respectively. Results: Hepatic leukemia factor (HLF) and E4 promoter binding protein-4 (E4BP4) regulated transcription of the mdr1a gene by competing with each other for the same DNA binding site. Molecular and biochemical analyses of HLF- and E4BP4-down-regulated colon 26 cells and the intestinal tract of Clock mutant mice suggested that these 2 proteins consisted of a reciprocating mechanism in which HLF activated the transcription of the mdr1a gene, whereas E4BP4 periodically suppressed transcription at the time of day when E4BP4 was abundant. Conclusions: The intestinal expression of the mdr1a gene is influenced by the circadian organization of molecular clockwork. Our present findings provide a link between the circadian timekeeping system and xenobiotic detoxification.

DOI： 10.1053/j.gastro.2008.07.073

Language：English  

OBJECTIVE: Although the pharmacokinetics of several drugs that are mainly eliminated by the CYP3A4 metabolism vary according to their dosing time, the mechanism of the variation remains poorly understood. In this study, we investigated how the 24-h oscillation in the expression of CYP3A4 mRNA was generated in hepatic cells. METHODS AND RESULTS: As brief exposure of HepG2 cells to 50&#37; serum induced the 24-h oscillation in the expression of clock genes, serum-shocked HepG2 cells were employed as an in-vitro model to study the molecular mechanism underlying the circadian clock in the human liver. Both mRNA levels and metabolic activity of CYP3A4 in serum-shocked HepG2 cells fluctuated rhythmically with a period length of about 24 h. The oscillation in the expression of the CYP3A4 gene seemed to be the underlying cause of the rhythmic change in its metabolic activity. Luciferase reporter gene analysis and electrophoretic mobility shift assay revealed that the circadian transcriptional factor, D-site-binding protein (DBP), activated the transcription of the CYP3A4 gene by binding to the DNA sequence near the upstream of the transcriptional start site. The transactivation of the CYP3A4 gene by DBP was repressed by the E4 promoter-binding protein-4 (E4BP4), a negative component of the circadian clock. CONCLUSIONS: Results from this study suggest that DBP and E4BP4 might consist of a reciprocating mechanism in which DBP activates the transcription of the CYP3A4 gene during the time of day when DBP is abundant, and E4BP4 suppresses the transcription at other times of day. Our current findings provide a molecular link between the circadian clock and the xenobiotic metabolism.

DOI： 10.1097/FPC.0b013e3282f12a61

Language：English  

Although the antiviral effect of interferon (IFN) varies depending on 24-h oscillation in the expression of its specific receptor, the mechanism of oscillation remains to be clarified. Here we report that oscillation in the expression of the IFN receptor gene (IFN-α/β R1) in mouse liver is caused by the endogenous rhythm of glucocorticoid secretion. Brief exposure of mouse hepatic cells (Hepa 1-6) to corticosterone (CORT) resulted in a significant decrease in mRNA levels of IFN-α/β R1. The CORT-induced decrease in IFN-α/β R1 mRNA levels was reversed by pretreating the cells with RU486, a glucocorticoid receptor antagonist. The mRNA levels of IFN-α/β R1 gene in the liver of adrenalectomized mice were consistently increased throughout the day. However, a single administration of CORT to adrenalectomized mice significantly decreased the mRNA levels of IFN-α/β R1 in the liver. Furthermore, the rhythmic phase of IFN-α/β R1 expression was modulated after the alteration of rhythmicity in glucocorticoid secretion, which was induced by restricted daily feeding. As a consequence, under manipulation of the feeding schedule, 2′-5′ oligoadenylate synthase activities, as an index of antiviral effect, in plasma and liver at 24 h after IFN-α injection also varied depending on the alteration of glucocorticoid secretion rhythm. These results suggest that the endogenous rhythm of glucocorticoid secretion is involved in the circadian regulation of IFN-α/β R1 expression in mouse liver. Our findings also support the notion that monitoring the 24-h variation in IFN receptor function is useful for selecting the most appropriate time of day to administer IFN.

DOI： 10.1210/en.2006-0415

Language：English  

Star fruit juice is a potent in vitro inhibitor of CYP3A; however, few reports are available on the inhibition of CYP3A activities by star fruit juice in vivo. Therefore, in this study, we investigated the CYP3A-mediated star fruit-drug interaction in vivo. The effect of star fruit juice on carbamazepine pharmacokinetics was examined in rats. In comparison with water, the area under the concentration-time curve (AUC) of carbamazepine was approximately 1.3-fold greater when star fruit juice (2 ml) was orally administered 1 h before the oral administration of carbamazepine (50 mg/kg). In contrast, the elimination half-life of carbamazepine and the AUC ratio of carbamazepine 10,11-epoxide to carbamazepine were not altered by the administration of star fruit juice. These results suggest that star fruit juice impairs the function of enteric CYP3A, but not of hepatic CYP3A. In addition, we evaluated the time course of recovery of CYP3A activity that was reduced after the treatment with star fruit juice. The inhibition by star fruit juice was recovered within approximately 24 h. These data suggest that the effect of star fruit juice is mainly reversible and transient. Thus, we discovered that star fruit juice alters the carbamazepine pharmacokinetics in rats.

DOI： 10.1124/dmd.105.006486

Language：English  

The effect of a typical neuroleptic haloperidol (Hal) on mPer1 gene expression was investigated in mouse suprachiasmatic nuclei (SCN). Hal induced mPer1 mRNA levels both in vivo and in cultured SCN cells. For mechanisms underlying Hal-induced mPer1 expression, N-methyl-D-aspartate (NMDA) glutamate receptor subtype, the phosphorylation form of the transcription factor, and the Ser-133 phosphorylation form of cAMP-responsive element-binding protein (CREB) played an important role, because the induction of mPer1 mRNA significantly decreased after pretreatment with a noncompetitive NMDA receptor antagonist, such as MK-801 or CREB antisense. These results suggest that Hal may increase CREB phosphorylation and mPer1 expression according to the activation of the NMDA receptor through the dopaminergic pathways. Although the injection of Hal during the light period increased the amplitude of mPer1 mRNA rhythmicity in a nondrug state, the injection of the drug during the dark period disturbed the rhythmic pattern of mPer1 mRNA. These results suggest that the rhythmicity of clock genes in SCN may be disturbed depending on the dosing time of Hal. On the other hand, because the induction of mPer1 mRNA by Hal seems to be at least partly caused by the NMDA receptor, showing a phase shift or resetting effect of the circadian clock, Hal may also cause such phase shift effects.

DOI： 10.1074/jbc.M411704200

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

The role of the susceptibility of cells and the pharmacokinetics of MTX on the time-dependent change of methotrexate (MTX) pharmacologic action in HL-60 (human leukemia cell) was investigated from the viewpoints of the rhythm of DNA synthesis. The highest activity of MTX was observed at the time when DNA synthesis, dihydrofolate reductase (DHFR) activity, DHFR content, and DHFR mRNA content increased and the lowest activity was observed at the time when they decreased. There were significant time-dependent changes in MTX efflux. The result corresponded to the rhythm in MTX activity. The present study suggests that the time-dependent change of MTX activity is caused by a change in the sensitivity of cells and the pharmacokinetics of the drug. Therefore, the choice of dosing time associated with cell rhythmicity may help to achieve rational chronotherapeutics, increasing therapeutic effects.

DOI： 10.1254/jphs.FP0050761

Language：English  

The influence of feeding schedule on the chronopharmacological aspects of acetaminophen (APAP) was examined in mice housed under 12-h light/dark cycle (lights on from 7:00 AM to 7:00 PM) with food and water ad libitum feeding (ALF) or under repeated time-restricted feeding (feeding time between 9:00 AM and 5:00 PM) for 2 weeks before the experiment. For the ALF group, there was a significant 24-h rhythm of mortality after APAP (600 mg/kg i.p.) injection. Peak mortality was observed after APAP injection at 9:00 PM and 1:00 AM, and nadir mortality was observed after drug injection at 9:00 AM. Hepatotoxicity after APAP (300 mg/kg i.p.) injection at 9:00 PM was significantly more severe than that after drug injection at 9:00 AM. Immunohistochemical staining using anti-APAP antibody 2 h after APAP injection was detected in centrilobular hepatocytes after drug injection at 9:00 PM but not after drug injection at 9:00 AM. CYP2E1 activity and hepatic glutathione (GSH) levels in untreated mice showed significant 24-h rhythms associated with APAP toxicity rhythm. The reduction in hepatic GSH levels after APAP injection at 9:00 PM was greater than that after drug injection at 9:00 AM. On the other hand, manipulation of the feeding schedule modified APAP hepatotoxicity rhythmicity, CYP2E1 activity, and GSH levels in the liver. Manipulation of the feeding schedule and choosing the most appropriate time of the day for drug administration may help to achieve rational chronopharmacology of some drugs including APAP in specific experimental and clinical situations.

DOI： 10.1124/jpet.104.069062

Language：English  

Purpose: Although the combination of Adriamycin (ADR) and docetaxel (DOC) showed a better cure rate against metastatic breast cancer in a clinical study, severe myelosuppression and cardiotoxicity were dose-limiting factors. The purpose of this study was to establish the most suitable dosing schedule to relieve severe adverse effects and improve the antitumor effects. Experimental Design: Both ADR and DOC were administered simultaneously in the simultaneous-dosing group (ADR/DOC), whereas in the intermittent-dosing groups (ADR-DOC and DOC-ADR), the second drug was administered 12 h after the first drug. Leukocyte counts and survival were measured to estimate adverse effects. After administration, ADR and DOC concentrations in blood, myelocyte cells, and heart were determined. To clarify the antitumor effect, tumor growth was measured in Ehrlich-cell-bearing mice after the initiation of drug injections. Results: The simultaneous-dosing group showed severe leukopenia compared with the saline-treated group. However, the toxicity was reduced in the intermittent-dosing groups. The DOC-ADR group showed the best survival rate in the dosing groups. In the pharmacokinetic study, ADR and DOC concentrations in plasma, myelocyte cells, and the heart were markedly higher in the simultaneous-dosing group than the intermittent-dosing groups. These results indicate that pharmacokinetic interactions may contribute to the change in leukopenia induced by concurrent administration of ADR and DOC. The antitumor effect in the DOC-ADR group was the highest in the dosing groups. Conclusions: In the present study, the findings suggest that ADR administered 12 h after DOC injection (DOC-ADR group) not only inhibits tumor growth more strongly but also significantly reduces leukopenia compared with results for the simultaneous-dosing (ADR/DOC) group and significantly reduced the number of toxic deaths compared with the other groups.

DOI： 10.1158/1078-0432.CCR-1000-03

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Event date： 2023.10 - 2023.11

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Venue：福岡   Country：Japan  

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Venue：福岡   Country：Japan  

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Venue：福岡   Country：Japan  

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Venue：札幌   Country：Japan  

Event date： 2023.3

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Venue：札幌   Country：Japan  

Event date： 2022.11 - 2022.12

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Venue：横浜   Country：Japan  

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Venue：横浜   Country：Japan  

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Venue：横浜   Country：Japan  

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Venue：横浜   Country：Japan  

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Event date： 2022.11

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Venue：長崎   Country：Japan  

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Venue：長崎   Country：Japan  

Event date： 2022.11

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Venue：長崎   Country：Japan  

Event date： 2022.10

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Venue：和歌山   Country：Japan  

Event date： 2022.9 - 2022.10

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Event date： 2022.3

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Venue：名古屋   Country：Japan  

Event date： 2021.12

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Venue：仙台   Country：Japan  

Event date： 2021.11

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Venue：熊本   Country：Japan  

Event date： 2021.7

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Venue：沖縄   Country：Japan  

Event date： 2020.12

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Venue：福岡   Country：Japan  

Event date： 2020.12

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Event date： 2020.12

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Venue：福岡   Country：Japan  

Event date： 2020.12

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Venue：福岡   Country：Japan  

Event date： 2020.12

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2020.12

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2020.12

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2020.12

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2019.11

Language：Japanese  

Venue：長崎   Country：Japan  

Event date： 2019.11

Language：Japanese  

Venue：長崎   Country：Japan  

Event date： 2019.10

Language：Japanese  

Venue：岐阜   Country：Japan  

Event date： 2018.11

Language：Japanese  

Country：Japan  

Event date： 2018.11

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2018.11

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2018.10

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：長崎大学   Country：Japan  

Event date： 2017.11

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：東京   Country：Japan  

Event date： 2017.7

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：鹿児島   Country：Japan  

Event date： 2016.12

Language：Japanese  

Venue：鹿児島   Country：Japan  

Event date： 2016.11

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2016.11

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2016.11

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2016.11

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2016.11

Language：Japanese  

Venue：群馬   Country：Japan  

Event date： 2015.5

Language：Japanese  

Venue：長崎   Country：Japan  

Event date： 2012.5

Language：Japanese  

Venue：神戸   Country：Japan  

Event date： 2011.11

Presentation type：Symposium, workshop panel (public)  

Venue：名古屋   Country：Japan  

Event date： 2011.5

Venue：東京   Country：Japan  

Event date： 2011.5

Venue：東京   Country：Japan  

Event date： 2010.12

Venue：長崎   Country：Japan  

Event date： 2010.11

Venue：東京   Country：Japan  

Event date： 2010.11

Venue：東京   Country：Japan  

Event date： 2010.9

Venue：大分   Country：Japan  

Event date： 2010.9

Venue：神戸   Country：Japan  

Event date： 2010.9

Venue：神戸   Country：Japan  

Event date： 2010.5

Venue：徳島   Country：Japan  

Event date： 2009.12

Venue：福岡   Country：Japan  

Event date： 2009.11

Venue：福岡   Country：Japan  

Event date： 2009.11

Venue：福岡   Country：Japan  

Event date： 2009.10

Venue：大阪   Country：Japan  

Event date： 2009.10

Venue：大阪   Country：Japan  

Event date： 2009.10

Venue：大阪   Country：Japan  

Event date： 2008.12

Venue：京都   Country：Japan  

Event date： 2008.12

Venue：京都）   Country：Japan  

Event date： 2008.12

Venue：京都   Country：Japan  

Event date： 2008.11

Venue：岡山）   Country：Japan  

Event date： 2008.11

Venue：岡山   Country：Japan  

Event date： 2008.10 - 2009.11

Venue：熊本   Country：Japan  

Event date： 2008.10 - 2008.11

Presentation type：Symposium, workshop panel (public)  

Venue：熊本   Country：Japan  

Event date： 2008.7

Venue：横浜国際会議場   Country：Cambodia  

Event date： 2008.5

Venue：札幌コンベンションセンター   Country：Japan  

Event date： 2008.5

Venue：札幌コンベンションセンター   Country：Japan  

Event date： 2002.12

Venue：福岡   Country：Japan  

Event date： 1998.12

Venue：福岡   Country：Japan  

Event date： 1998.5

Venue：神戸   Country：Japan  

Event date： 1997.12

Venue：東京   Country：Japan  

Event date： 1997.3

Venue：町田   Country：Japan  

Language：Japanese  

Venue：福岡市   Country：Japan  

Language：Japanese  

Venue：福岡市   Country：Japan  

Language：Japanese  

Venue：松山市   Country：Japan  

Language：Japanese  

Venue：福岡市   Country：Japan  

Language：Japanese  

Venue：名古屋市   Country：Japan  

Language：Japanese  

Venue：福岡市   Country：Japan  

Venue：大分   Country：Japan  

Venue：福岡   Country：Japan  

Venue：大阪   Country：Japan  

Venue：大阪   Country：Japan  

Venue：大分   Country：Japan  

Venue：長崎   Country：Japan  

Venue：福岡市   Country：Japan  

Venue：福岡市   Country：Japan  

Venue：福岡市   Country：Japan  

Venue：埼玉   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：北海道   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：京都   Country：Japan  

Language：Japanese  

Venue：京都   Country：Japan  

Language：Japanese  

Venue：熊本   Country：Japan  

Language：Japanese  

Venue：熊本   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：名古屋   Country：Japan  

Language：Japanese  

Venue：名古屋   Country：Japan  

Language：Japanese  

Venue：名古屋   Country：Japan  

Language：Japanese  

Venue：名古屋   Country：Japan  

Language：Japanese  

Venue：大阪   Country：Japan  

Language：Japanese  

Venue：大阪   Country：Japan  

Language：Japanese  

Venue：大阪   Country：Japan  

Language：Japanese  

Venue：仙台   Country：Japan  

Language：Japanese  

Venue：仙台   Country：Japan  

Language：Japanese  

Venue：長崎   Country：Japan  

Language：Japanese  

Venue：長崎   Country：Japan  

Language：Japanese  

Venue：横浜   Country：Japan  

Language：Japanese  

Venue：名古屋   Country：Japan  

Language：Japanese  

Venue：福岡市   Country：Japan  

Event date： 2023.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Event date： 2023.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌   Country：Japan  

Event date： 2022.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：横浜   Country：Japan  

Event date： 2021.11

Language：Japanese  

Venue：群馬   Country：Japan  

Event date： 2021.11

Language：Japanese  

Venue：群馬   Country：Japan  

Event date： 2021.11

Language：Japanese  

Venue：群馬   Country：Japan  

Event date： 2021.11

Language：Japanese  

Venue：熊本   Country：Japan  

Event date： 2021.10

Language：Japanese  

Venue：横浜   Country：Japan  

Event date： 2021.10

Language：Japanese  

Venue：東京   Country：Japan  

Event date： 2021.7

Language：Japanese  

Venue：沖縄   Country：Japan  

Event date： 2021.5

Language：Japanese  

Venue：徳島   Country：Japan  

Event date： 2021.3

Language：Japanese  

Venue：広島   Country：Japan  

Event date： 2020.12

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2020.12

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2019.11

Language：Japanese  

Venue：長崎   Country：Japan  

Event date： 2019.11

Language：Japanese  

Venue：長崎   Country：Japan  

Event date： 2019.11

Language：Japanese  

Venue：長崎   Country：Japan  

Event date： 2019.10

Language：Japanese  

Venue：岐阜   Country：Japan  

Event date： 2018.12

Language：Japanese  

Country：Japan  

Event date： 2018.11

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2018.11

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2018.11

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2018.10 - 2018.12

Language：Japanese  

Country：Japan  

Event date： 2018.10

Language：Japanese  

Country：Japan  

Event date： 2018.6

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

Event date： 2018.6

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2018.6

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2017.12

Language：Japanese  

Venue：熊本   Country：Japan  

Event date： 2017.12

Language：Japanese  

Venue：熊本   Country：Japan  

Event date： 2017.12

Language：Japanese  

Venue：熊本   Country：Japan  

Event date： 2017.12

Language：Japanese  

Venue：熊本   Country：Japan  

Event date： 2017.10

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2017.10

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2017.8

Language：Japanese  

Venue：大分   Country：Japan  

Event date： 2017.6

Language：Japanese  

Venue：沖縄   Country：Japan  

Event date： 2017.4

Language：Japanese  

Venue：東京   Country：Japan  

Event date： 2016.12

Language：Japanese  

Venue：鹿児島   Country：Japan  

Event date： 2016.12

Language：Japanese  

Venue：鹿児島   Country：Japan  

Event date： 2016.12

Language：Japanese  

Venue：鹿児島   Country：Japan  

Event date： 2016.12

Language：Japanese  

Venue：鹿児島   Country：Japan  

Event date： 2016.11

Language：Japanese  

Venue：松山市   Country：Japan  

Event date： 2016.11

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2016.11

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2016.11

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2016.11

Language：Japanese  

Venue：群馬   Country：Japan  

Event date： 2016.9

Language：English   Presentation type：Oral presentation (general)  

Venue：Taipei   Country：Taiwan, Province of China  

Event date： 2016.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：kyoto   Country：Japan  

Event date： 2016.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：kyoto   Country：Japan  

Event date： 2016.6

Language：English  

Venue：Busan   Country：Korea, Republic of  

Event date： 2016.5

Language：English  

Venue：岐阜市   Country：Japan  

Event date： 2016.4

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2016.3

Language：Japanese  

Venue：横浜   Country：Japan  

Event date： 2016.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Event date： 2016.3

Language：Japanese  

Venue：横浜   Country：Japan  

Event date： 2016.3

Language：Japanese  

Venue：横浜   Country：Japan  

Event date： 2016.3

Language：Japanese  

Venue：横浜   Country：Japan  

Event date： 2016.3

Language：Japanese  

Venue：横浜   Country：Japan  

Event date： 2015.11

Language：Japanese  

Venue：千葉市   Country：Japan  

Event date： 2015.11

Language：Japanese  

Venue：千葉市   Country：Japan  

Event date： 2012.5

Language：Japanese  

Venue：神戸   Country：Japan  

Event date： 2012.5

Language：Japanese  

Venue：神戸   Country：Japan  

Event date： 2011.12

Venue：福岡   Country：Japan  

Event date： 2011.12

Venue：福岡   Country：Japan  

Event date： 2011.12

Venue：福岡   Country：Japan  

Event date： 2011.12

Venue：福岡   Country：Japan  

Event date： 2011.12

Venue：福岡   Country：Japan  

Event date： 2011.11

Venue：名古屋   Country：Japan  

Event date： 2011.11

Venue：名古屋   Country：Japan  

Event date： 2011.11

Venue：名古屋   Country：Japan  

Event date： 2011.11

Venue：名古屋   Country：Japan  

Event date： 2011.11

Venue：名古屋   Country：Japan  

Event date： 2011.11

Venue：名古屋   Country：Japan  

Event date： 2011.11

Venue：名古屋   Country：Japan  

Event date： 2011.11

Venue：名古屋   Country：Japan  

Event date： 2011.11

Venue：名古屋   Country：Japan  

Event date： 2011.7

Venue：福岡   Country：Japan  

Event date： 2011.7

Venue：旭川   Country：Japan  

Event date： 2011.7

Venue：旭川   Country：Japan  

Event date： 2010.10

Venue：大阪   Country：Japan  

Event date： 2010.10

Venue：大阪   Country：Japan  

Event date： 2010.10

Venue：Fukuoka   Country：Japan  

Event date： 2010.9

Venue：神戸   Country：Japan  

Event date： 2009.12 - 2010.5

Venue：福岡   Country：Japan  

Event date： 2009.12

Venue：(福岡)   Country：Japan  

Event date： 2009.12 - 2009.5

Venue：福岡   Country：Japan  

Event date： 2009.11

Venue：福岡   Country：Japan  

Event date： 2009.11

Venue：Tukuba   Country：Japan  

Event date： 2009.10

Venue：大阪   Country：Japan  

Event date： 2009.10

Venue：大阪   Country：Japan  

Event date： 2009.10

Venue：大阪   Country：Japan  

Event date： 2009.5

Venue：静岡   Country：Japan  

Event date： 2009.3

Venue：京都   Country：Japan  

Event date： 2008.11

Venue：岡山   Country：Japan  

Event date： 2008.10 - 2008.11

Venue：熊本   Country：Japan  

Event date： 2008.10 - 2008.11

Venue：熊本   Country：Japan  

Event date： 2008.10 - 2008.11

Venue：熊本   Country：Japan  

Event date： 2008.5

Venue：札幌コンベンションセンター   Country：Japan  

Event date： 2002.5

Language：Japanese  

Venue：神戸   Country：Japan  

Event date： 1997.11

Venue：久留米   Country：Japan  

Language：Japanese  

Venue：福岡市   Country：Japan  

Language：Japanese  

Venue：福岡市   Country：Japan  

Language：Japanese  

Venue：福岡市   Country：Japan  

Language：Japanese  

Venue：福岡市   Country：Japan  

Language：Japanese  

Venue：福岡市   Country：Japan  

Language：Japanese  

Venue：福岡市   Country：Japan  

Venue：鹿児島   Country：Japan  

Venue：岡山   Country：Japan  

Venue：仙台   Country：Japan  

Venue：熊本   Country：Japan  

Venue：熊本   Country：Japan  

Venue：熊本   Country：Japan  

Venue：埼玉   Country：Japan  

Venue：埼玉   Country：Japan  

Venue：福岡市   Country：Japan  

Venue：福岡市   Country：Japan  

Venue：埼玉   Country：Japan  

Venue：福岡市   Country：Japan  

Language：Japanese  

Venue：大阪市   Country：Japan  

Language：Japanese  

Venue：大阪市   Country：Japan  

Language：Japanese  

Venue：大阪市   Country：Japan  

Language：Japanese  

Venue：大阪市   Country：Japan  

Language：Japanese  

Venue：大阪市   Country：Japan  

Language：Japanese  

Venue：大阪市   Country：Japan  

Language：Japanese  

Venue：大阪市   Country：Japan  

Language：Japanese  

Venue：大阪市   Country：Japan  

Language：Japanese  

Venue：大阪市   Country：Japan  

Language：Japanese  

Venue：福岡市   Country：Japan  

Language：Japanese  

Venue：京都市   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：大阪   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：北海道   Country：Japan  

Language：Japanese  

Venue：熊本   Country：Japan  

Language：Japanese  

Venue：熊本   Country：Japan  

Language：Japanese  

Venue：熊本   Country：Japan  

Language：Japanese  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：大阪市   Country：Japan