Circadian rhythms in CYP2A5 expression underlie the time-dependent effect of tegafur on breast cancer.

The chemotherapeutic agent tegafur, a prodrug that prolongs the half-life of fluorouracil (5-FU), exerts antitumor effects against various cancers. Since tegafur is metabolized to 5-FU by CYP2A6 in the liver, the expression of CYP2A6 determines the effect of tegafur. Here, we report that the expression rhythm of Cyp2a5, a homolog of human CYP2A6, in female mice causes dosing time-dependent differences in tegafur metabolism. In the livers of female mice, CYP2A5 expression showed a circadian rhythm, peaking during the dark period. This rhythm is regulated by RORA, a core clock component, and abrogation of the CYP2A5 activity abolished the time-dependent difference in the rate of tegafur metabolism in female mice. Furthermore, administration of tegafur to mice transplanted with 4T1 breast cancer cells during the dark period suppressed increases in tumor size compared to female mice treated during the light period. Our findings reveal a novel relationship between 5-FU prodrugs and circadian clock machinery, potentially influencing antitumor effects, and contributing to the development of time-aware chemotherapy regimens for breast cancer.

Hericenone C attenuates the second phase of formalin-induced nociceptive behavior by suppressing the accumulation of CD11c-positive cells in the paw epidermis via phosphorylated P65.

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.

Time-Dependent Differences in Vancomycin Sensitivity of Macrophages Underlie Vancomycin-Induced Acute Kidney Injury.

Although vancomycin (VCM)-frequently used to treat drug-resistant bacterial infections-often induces acute kidney injury (AKI), discontinuation of the drug is the only effective treatment; therefore, analysis of effective avoidance methods is urgently needed. Here, we report the differences in the induction of AKI by VCM in 1/2-nephrectomized mice depending on the time of administration. Despite the lack of difference in the accumulation of VCM in the kidney between the light (ZT2) and dark (ZT14) phases, the expression of AKI markers due to VCM was observed only in the ZT2 treatment. Genomic analysis of the kidney suggested that the time of administration was involved in VCM-induced changes in monocyte and macrophage activity, and VCM had time-dependent effects on renal macrophage abundance, ATP activity, and interleukin (IL)-1ß expression. Furthermore, the depletion of macrophages with clodronate abolished the induction of IL-1ß and AKI marker expression by VCM administration at ZT2. This study provides evidence of the need for time-dependent pharmacodynamic considerations in the prevention of VCM-induced AKI as well as the potential for macrophage-targeted AKI therapy. SIGNIFICANCE STATEMENT: There is a time of administration at which vancomycin (VCM)-induced renal injury is more and less likely to occur, and macrophages are involved in this difference. Therefore, there is a need for time-dependent pharmacodynamic considerations in the prevention of VCM-induced acute kidney injury as well as the potential for macrophage-targeted acute kidney injury therapy.

Age-Related Effects on MSC Immunomodulation, Macrophage Polarization, Apoptosis, and Bone Regeneration Correlate with IL-38 Expression.

Mesenchymal stem cells (MSCs) are known to promote tissue regeneration and suppress excessive inflammation caused by infection or trauma. Reported evidence indicates that various factors influence the expression of MSCs' endogenous immunomodulatory properties. However, the detailed interactions of MSCs with macrophages, which are key cells involved in tissue repair, and their regulatory mechanisms are not completely understood. We herein investigated how age-related immunomodulatory impairment of MSCs alters the interaction of MSCs with macrophages during bone healing using young (5-week old) and aged (50-week old) mice. To clarify the relationship between inflammatory macrophages (M1) and MSCs, their spatiotemporal localization at the bone healing site was investigated by immunostaining, and possible regulatory mechanisms were analyzed in vitro co-cultures. Histomorphometric analysis revealed an accumulation of M1 and a decrease in MSC number at the healing site in aged mice, which showed a delayed bone healing. In in vitro co-cultures, MSCs induced M1 apoptosis through cell-to-cell contact but suppressed the gene expression of pro-inflammatory cytokines by soluble factors secreted in the culture supernatant. Interestingly, interleukin 38 (Il-38) expression was up-regulated in M1 after co-culture with MSCs. IL-38 suppressed the gene expression of inflammatory cytokines in M1 and promoted the expression of genes associated with M1 polarization to anti-inflammatory macrophages (M2). IL-38 also had an inhibitory effect on M1 apoptosis. These results suggest that MSCs may induce M1 apoptosis, suppress inflammatory cytokine production by M1, and induce their polarization toward M2. Nevertheless, in aged conditions, the decreased number and immunomodulatory function of MSCs could be associated with a delayed M1 clearance (i.e., apoptosis and/or polarization) and consequent delayed resolution of the inflammatory phase. Furthermore, M1-derived IL-38 may be associated with immunoregulation in the tissue regeneration site.

Hematopoietic Prostaglandin D Synthase Is Increased in Mast Cells and Pericytes in Autopsy Myocardial Specimens from Patients with Duchenne Muscular Dystrophy.

The leading cause of death for patients with Duchenne muscular dystrophy (DMD), a progressive muscle disease, is heart failure. Prostaglandin (PG) D2, a physiologically active fatty acid, is synthesized from the precursor PGH2 by hematopoietic prostaglandin D synthase (HPGDS). Using a DMD animal model (mdx mice), we previously found that HPGDS expression is increased not only in injured muscle but also in the heart. Moreover, HPGDS inhibitors can slow the progression of muscle injury and cardiomyopathy. However, the location of HPGDS in the heart is still unknown. Thus, this study investigated HPGDS expression in autopsy myocardial samples from DMD patients. We confirmed the presence of fibrosis, a characteristic phenotype of DMD, in the autopsy myocardial sections. Additionally, HPGDS was expressed in mast cells, pericytes, and myeloid cells of the myocardial specimens but not in the myocardium. Compared with the non-DMD group, the DMD group showed increased HPGDS expression in mast cells and pericytes. Our findings confirm the possibility of using HPGDS inhibitor therapy to suppress PGD2 production to treat skeletal muscle disorders and cardiomyopathy. It thus provides significant insights for developing therapeutic drugs for DMD.

Prostaglandin F2α Affects the Cycle of Clock Gene Expression and Mouse Behavior.

Prostaglandins are bioactive compounds, and the activation of their receptors affects the expression of clock genes. However, the prostaglandin F receptor (Ptgfr) has no known relationship with biological rhythms. Here, we first measured the locomotor period lengths of Ptgfr-KO (B6.129-Ptgfrtm1Sna) mice and found that they were longer under constant dark conditions (DD) than those of wild-type (C57BL/6J) mice. We then investigated the clock gene patterns within the suprachiasmatic nucleus in Ptgfr-KO mice under DD and observed a decrease in the expression of the clock gene cryptochrome 1 (Cry1), which is related to the circadian cycle. Moreover, the expression of Cry1, Cry2, and Period2 (Per2) mRNA were significantly altered in the mouse liver in Ptgfr-KO mice under DD. In the wild-type mouse, the plasma prostaglandin F2α (PGF2α) levels showed a circadian rhythm under a 12 h cycle of light-dark conditions. In addition, in vitro experiments showed that the addition of PTGFR agonists altered the amplitude of Per2::luc activity, and this alteration differed with the timing of the agonist addition. These results lead us to hypothesize that the plasma rhythm of PGF2α is important for driving clock genes, thus suggesting the involvement of PGF2α- and Ptgfr-targeting drugs in the biological clock cycle.

Inhibition of Tumor-Derived C-C Motif Chemokine Ligand 2 Expression Attenuates Tactile Allodynia in NCTC 2472 Fibrosarcoma-Inoculated Mice.

Neuropathic pain associated with cancers is caused by tumor growth compressing and damaging nerves, which would also be enhanced by inflammatory factors through sensitizing nociceptor neurons. A troublesome hallmark symptom of neuropathic pain is hypersensitivity to innocuous stimuli, a condition known as "tactile allodynia", which is often refractory to NSAIDs and opioids. The involvement of chemokine CCL2 (monocyte chemoattractant protein-1) in cancer-evoked neuropathic pain is well established, but opinions remain divided as to whether CCL2 is involved in the production of tactile allodynia with tumor growth. In this study, we constructed Ccl2 knockout NCTC 2472 (Ccl2-KO NCTC) fibrosarcoma cells and conducted pain behavioral test using Ccl2-KO NCTC-implanted mice. Implantation of naïve NCTC cells around the sciatic nerves of mice produced tactile allodynia in the inoculated paw. Although the growth of Ccl2 KO NCTC-formed tumors was comparable to that of naïve NCTC-formed tumors, Ccl2-KO NCTC-bearing mice failed to show tactile pain hypersensitivity, suggesting the involvement of CCL2 in cancer-induced allodynia. Subcutaneous administration of controlled-release nanoparticles containing the CCL2 expression inhibitor NS-3-008 (1-benzyl-3-hexylguanidine) significantly attenuated tactile allodynia in naïve NCTC-bearing mice accompanied by a reduction of CCL2 content in tumor masses. Our present findings suggest that inhibition of CCL2 expression in cancer cells is a useful strategy to attenuate tactile allodynia induced by tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for the treatment of cancer-evoked neuropathic pain. SIGNIFICANCE STATEMENT: The blockade of chemokine/receptor signaling, particularly for C-C motif chemokine ligand 2 (CCL2) and its high-affinity receptor C-C chemokine receptor type 2 (CCR2), has been implicated to attenuate cancer-induced inflammatory and nociceptive pain. This study demonstrated that continuous inhibition of CCL2 production from cancer cells also prevents the development of tactile allodynia associated with tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for management of cancer-evoked tactile allodynia.

RNA editing enzyme ADAR1 controls miR-381-3p-mediated expression of multidrug resistance protein MRP4 via regulation of circRNA in human renal cells.

Multidrug resistance-associated protein 4 (MRP4), a member of the C subfamily of ATP-binding cassette transporters, is highly expressed in the kidneys of mammals and is responsible for renal elimination of numerous drugs. Adenosine deaminase acting on RNA 1 (ADAR1) has been reported to regulate gene expression by catalyzing adenosine-to-inosine RNA editing reactions; however, potential roles of ADAR1 in the regulation of MRP4 expression have not been investigated. In this study, we found that downregulation of ADAR1 increased the expression of MRP4 in human renal cells at the posttranscriptional level. Luciferase reporter assays and microarray analysis revealed that downregulation of ADAR1 reduced the levels of microRNA miR-381-3p, which led to the corresponding upregulation of MPR4 expression. Circular RNAs (circRNAs) are a type of closed-loop endogenous noncoding RNAs that play an essential role in gene expression by acting as miRNA sponges. We demonstrate that ADAR1 repressed the biogenesis of circRNA circHIPK3 through its adenosine-to-inosine RNA editing activity, which altered the secondary structure of the precursor of circHIPK3. Furthermore, in silico analysis suggested that circHIPK3 acts as a sponge of miR-381-3p. Indeed, we found overexpression of circHIPK3 induced the expression of MRP4 through its interference with miR-381-3p. Taken together, our study provides novel insights into regulation of the expression of xenobiotic transporters through circRNA expression by the RNA editing enzyme ADAR1.

Fluorescence-Based Detection of Fatty Acid ß-Oxidation in Cells and Tissues Using Quinone Methide-Releasing Probes.

Detection of metabolic activity enables us to reveal the inherent metabolic state of cells and elucidate mechanisms underlying cellular homeostasis and growth. However, a fluorescence approach for the study of metabolic pathways is still largely unexplored. Herein, we have developed a new chemical probe for the fluorescence-based detection of fatty acid ß-oxidation (FAO), a key process in lipid catabolism, in cells and tissues. This probe serves as a substrate of FAO and forms a reactive quinone methide (QM) as a result of metabolic reactions. The liberated QM is covalently captured by intracellular proteins, and subsequent bio-orthogonal ligation with a fluorophore enables fluorescence analysis. This reaction-based sensing allowed us to detect FAO activity in cells at a desired emission wavelength using diverse analytical techniques including fluorescence imaging, in-gel fluorescence activity-based protein profiling (ABPP), and fluorescence-activated cell sorting (FACS). The probe was able to detect changes in FAO activity induced by chemical modulators in cultured cells. The probe was further employed for fluorescence imaging of FAO in mouse liver tissues and revealed the metabolic heterogeneity of FAO activity in hepatocytes by the combination of FACS and gene expression analysis, highlighting the utility of our probe as a chemical tool for fatty acid metabolism research.

Oral administration of vancomycin alleviates heart failure triggered by chronic kidney disease.

Chronic kidney disease (CKD) induces an imbalance in the intestinal microbiota, affecting various physiological functions and leading to cardiovascular inflammation and fibrosis. However, the cardiotoxic impact of intestinal microbiota-derived uremic substances in advanced renal dysfunction remains unexplored. Therefore, we developed a 5/6 nephrectomy (5/6Nx) mouse model to investigate the intestinal microbiota and the effects of administering vancomycin (VCM) on the microbiota and the cardiac pathology associated with CKD. Despite VCM administration after the development of irreversible glomerulosclerosis and tubulointerstitial fibrosis, blood indoxyl sulfate and phenyl sulfate levels, which are intestinal bacteria-derived uremic substances, brain natriuretic peptide levels, and the fibrotic area in the heart were decreased. Moreover, VCM administration prevented 5/6Nx-induced weight loss and prolonged survival time. Our findings suggest that VCM-induced changes in the intestinal microbiota composition ameliorate heart failure and improve survival rates by reducing intestinal microbiota-derived cardiotoxic substances despite advanced renal dysfunction. This highlights the potential of using the intestinal microbiota as a target to prevent and treat cardiovascular conditions associated with CKD.

Population Pharmacokinetic Analysis of Drug-Drug Interactions Between Perampanel and Carbamazepine Using Enzyme Induction Model in Epileptic Patients.

BACKGROUND: Perampanel (PER) is an oral antiepileptic drug and its concomitant use with carbamazepine (CBZ) leads to decreased PER concentrations. However, the magnitude of its influence may vary, depending on the dynamics of the enzyme induction properties of CBZ. This study aimed to develop a population pharmacokinetic (PPK) model considering the dynamics of enzyme induction and evaluate the effect of CBZ on PER pharmacokinetics. METHODS: We retrospectively collected data on patient background, laboratory tests, and prescribed drugs from electronic medical records. We developed 2 PPK models incorporating the effect of CBZ-mediated enzyme induction to describe time-concentration profiles of PER using the following different approaches: (1) treating the concomitant use of CBZ as a categorical covariate (empirical PPK model) and (2) incorporating the time-course of changes in the amount of enzyme by CBZ-mediated induction (semimechanistic PPK model). The bias and precision of the predictions were investigated by calculating the mean error, mean absolute error, and root mean squared error. RESULTS: A total of 133 PER concentrations from 64 patients were available for PPK modelling. PPK analyses showed that the co-administration of CBZ increased the clearance of PER. Goodness-of-fit plots indicated a favorable description of the observed data and low bias. The mean error, mean absolute error, and root mean square error values based on the semimechanistic model were smaller than those obtained using the empirical PPK model for predicting PER concentrations in patients with CBZ. CONCLUSIONS: We developed 2 PPK models to describe PER pharmacokinetics based on different approaches, using electronic medical record data. Our PPK models support the use of PER in clinical practice.

RNA editing enzyme ADAR1 governs the circadian expression of P-glycoprotein in human renal cells by regulating alternative splicing of the ABCB1 gene.

The expression and function of some xenobiotic transporters vary according to the time of the day, causing the dosing time-dependent changes in drug disposition and toxicity. P-glycoprotein (P-gp), encoded by the ABCB1 gene, is highly expressed in the kidneys and functions in the renal elimination of various drugs. The elimination of several P-gp substrates was demonstrated to vary depending on administration time, but the underlying mechanism remains unclear. We found that adenosine deaminase acting on RNA (ADAR1) was involved in the circadian regulation of P-gp expression in human renal proximal tubular epithelial cells (RPTECs). After synchronization of the cellular circadian clock by dexamethasone treatment, the expression of P-gp exhibited a significant 24-h oscillation in RPTECs, but this oscillation was disrupted by the downregulation of ADAR1. Although ADAR1 catalyzes adenosine-to-inosine (A-to-I) RNA editing in double-stranded RNA substrates, no significant ADAR1-regulated editing sites were detected in the human ABCB1 transcripts in RPTECs. On the other hand, downregulation of ADAR1 induced alternative splicing in intron 27 of the human ABCB1 gene, resulting in the production of retained intron transcripts. The aberrant spliced transcript was sensitive to nonsense-mediated mRNA decay, leading to the decreased stability of ABCB1 mRNA and prevention of the 24-h oscillation of P-gp expression. These findings support the notion that ADAR1-mediated regulation of alternative splicing of the ABCB1 gene is a key mechanism of circadian expression of P-gp in RPTECs, and the regulatory mechanism may underlie the dosing time-dependent variations in the renal elimination of P-gp substrates.

Chronopharmacology of immune-related diseases.

Clock genes, circadian pacemaker resides in the paired suprachiasmatic nuclei (SCN), control various circadian rhythms in many biological processes such as physiology and behavior. Clock gene regulates many diseases such as cancer, immunological dysfunction, metabolic syndrome and sleep disorders etc. Chronotherapy is especially relevant, when the risk and/or intensity of the symptoms of disease vary predicably over time as exemplified by allergic rhinitis, arthritis, asthma, myocardial infarction, congestive heart failure, stroke, and peptic ulcer disease. Dosing time influences the effectiveness and toxicity of many drugs. The pharmacodynamics of medications as well as pharmacokinetics influences chronopharmacological phenomena. To escape from host immunity in the tumor microenvironment, cancer cells have acquired several pathways. Immune checkpoint therapy targeting programmed death 1 (PD-1) and its ligand (PD-L1) interaction had been approved for the treatment of patients with several types of cancers. Circadian expression of PD-1 is identified on tumor associated macrophages (TAMs), which is rationale for selecting the most appropriate time of day for administration of PD-1/PD-L1 inhibitors. The therapies for chronic kidney disease (CKD) are urgently needed because of a global health problem. The mechanism of the cardiac complications in mice with CKD had been related the GRP68 in circulating monocytes and serum accumulation of retinol. Development of a strategy to suppress retinol accumulation will be useful to prevent the cardiac complications of CKD. Therefore, we introduce an overview of the dosing time-dependent changes in therapeutic outcome and safety of drug for immune-related diseases.

Circadian expression of Glycoprotein 2 (Gp2) gene is controlled by a molecular clock in mouse Peyer's patches.

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.

Selective and reversible modification of kinase cysteines with chlorofluoroacetamides.

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1-10 µM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton's tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.

Mutation of the gene encoding the circadian clock component PERIOD2 in oncogenic cells confers chemoresistance by up-regulating the Aldh3a1 gene.

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 (Per2m/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-rasV12 and SV40 large T-antigen induced malignant transformation of both WT and Per2m/m cells, but the cytotoxic effects of the chemotherapeutic agents methotrexate, gemcitabine, etoposide, vincristine, and oxaliplatin were significantly alleviated in the oncogene-transformed Per2m/m cells. Although introduction of the two oncogenes increased the expression of Aldh3a1 in both WT and Per2m/m cells, the ALDH3A1 protein levels in the Per2m/m cells were ∼7-fold higher than in WT cells. The elevated ALDH3A1 levels in the oncogene-transformed Per2m/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 Per2m/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.

Bicyclobutane Carboxylic Amide as a Cysteine-Directed Strained Electrophile for Selective Targeting of Proteins.

Expanding the repertoire of electrophiles with unique reactivity features would facilitate the development of covalent inhibitors with desirable reactivity profiles. We herein introduce bicyclo[1.1.0]butane (BCB) carboxylic amide as a new class of thiol-reactive electrophiles for selective and irreversible inhibition of targeted proteins. We first streamlined the synthetic routes to generate a variety of BCB amides. The strain-driven nucleophilic addition to BCB amides proceeded chemoselectively with cysteine thiols under neutral aqueous conditions, the rate of which was significantly slower than that of acrylamide. This reactivity profile of BCB amide was successfully exploited to develop covalent ligands targeting Bruton's tyrosine kinase (BTK). By tuning BCB amide reactivity and optimizing its disposition on the ligand, we obtained a selective covalent inhibitor of BTK. The in-gel activity-based protein profiling and mass spectrometry-based chemical proteomics revealed that the selected BCB amide had a higher target selectivity for BTK in human cells than did a Michael acceptor probe. Further chemical proteomic study revealed that BTK probes bearing different classes of electrophiles exhibited distinct off-target profiles. This result suggests that incorporation of BCB amide as a cysteine-directed electrophile could expand the capability to develop covalent inhibitors with the desired proteome reactivity profile.

Microcurrent stimulation activates the circadian machinery in mice.

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.

Estradiol regulation of P-glycoprotein expression in mouse kidney and human tubular epithelial cells, implication for renal clearance of drugs.

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 17ß-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 17ß-estradiol, but not testosterone. Similar 17ß-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.

Dosing Time-Dependent Changes in the Anti-tumor Effect of xCT Inhibitor Erastin in Human Breast Cancer Xenograft Mice.

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.