Published papers

Composition and inducibility of cytochrome P450 (CYP) in ocular tissues were investigated using reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot techniques. Composition of ocular CYPs is more restricted than that of hepatic forms. High levels of CYP2B1/2 and reduced levels of CYP2C11 expression were detected in rat ocular tissues. Phenobarbital induced CYP2B1/2 expression in the lens but not in the rest of the eye, whereas CYP2C11 induction was observed in both regions. In addition, CYP2B1/2 proteins were found to be prominent in the retina. Our data indicate eye-specific regulation of CYP expression.

To explore the physiological roles of cytochrome P450 (CYP) in peripheral blood cells, we examined which isoforms of CYP families were expressed in human myeloid leukemia cell lines (U937, HL-60 and K562) and lymphoid cell lines (BALL-1, MOLT-4 and Jurkat) by RT-PCR. We observed relatively high expression of CYP1A1, CYP1B1, CYP2A6, CYP2A7, CYP2D6, and CYP2E1 in all cell types, but CYP2A13 and CYP2C9 expression was not detected. Expressions of aryl hydrocarbon (Ah) receptor and Ah receptor nuclear translocator (ARNT), which mediate induction of the CYP1 family, were also detected in all cell types. Cell-type specific expression of CYP3A4 and CYP3A5 was observed in MOLT-4 and K562 cells. Weak, but significant, expression of CYP3A7 was detected in K562 cells. The profile of CYP expression in the culture cells reported here provides information that furthers our understanding of the physiological roles of CYP enzymes in human blood cells.

Recent epidemiological studies have shown that moderate coffee consumption is associated with a lower risk of certain types of cancers, particularly colon cancer in postmenopausal women. To elucidate the molecular basis for the preventive action of coffee, we investigated the effect of coffee on estrogen sulfotransferase (SULT) because sulfation is the major pathway involved in the inactivation of estrogens. We found that coffee reduced SULT1E1 gene expression in human colon carcinoma Caco-2 cells. Treatment with 2.5% (v/v) coffee for 24 h resulted in a 60% reduction of the expression of the SULT1E1 gene in Caco-2 cells. Corresponding to reduced SULT1E1 gene expression, cytosolic estrogen SULT activity toward E2 (20 nm) decreased by 25%. In addition, an accumulation of E2 sulfates in the medium, which reflects cellular activity of estrogen SULT, decreased after the cells were treated with coffee. Major bioactive constituents in coffee such as caffeine, chlorogenic acid and caffeic acid did not show any effect. The inhibitory activity was extractable by using ethyl acetate. We also found that the inhibitory activity was produced by roasting the coffee beans. Mithramycin, an inhibitor of the transcription factor stimulating protein 1 (Sp1), was able to restore coffee-reduced SULT1E1 gene expression. Our data suggest that daily coffee consumption may reduce estrogen SULT activity, thereby enhancing estrogenic activity in the colon. © 2013 The Pharmaceutical Society of Japan.

Coffee is a beverage that is consumed world-wide on a daily basis and is known to induce a series of metabolic and pharmacological effects, especially in the digestive tract. However, little is known concerning the effects of coffee on transporters in the gastrointestinal tract. To elucidate the effect of coffee on intestinal transporters, we investigated its effect on expression of the breast cancer resistance protein (BCRP/ABCG2) in a human colorectal cancer cell line, Caco-2. Coffee induced BCRP gene expression in Caco-2 cells in a coffee-dose dependent manner. Coffee treatment of Caco-2 cells also increased the level of BCRP protein, which corresponded to induction of gene expression, and also increased cellular efflux activity, as judged by Hoechst33342 accumulation. None of the major constituents of coffee tested could induce BCRP gene expression. The constituent of coffee that mediated this induction was extractable with ethyl acetate and was produced during the roasting process. Dehydromethylepoxyquinomicin (DHMEQ), an inhibitor of nuclear factor (NF)-κB, inhibited coffee-mediated induction of BCRP gene expression, suggesting involvement of NF-κB in this induction. Our data suggest that daily consumption of coffee might induce BCRP expression in the gastrointestinal tract and may affect the bioavailability of BCRP substrates. © 2011 Pharmaceutical Society of Japan.

The placenta acts as a barrier that protects the fetus from xenobiotics in the maternal blood. Phase II conjugation reactions in the placenta are considered to play an important role in this protective process by transferring polar hydroxyl groups and thereby imparting water solubility that facilitates the excretion of xenobiotics. Since coffee consumption during pregnancy is reported to affect fetal growth and development, we examined its effects upon the conjugation reactions in the rat syncytiotrophoblast cell line TR-TBT 18d-1. We detected a high level of glucuronidation for 1-naphthol, a model compound, in both a dose- and culture time-dependent manner in the TR-TBT 18d-1 cells. However, no sulfation was detected in any of our analyses. Coffee was found to inhibit the glucuronidation of 1-naphthol with an IC50 of 4.5% (v/v). In contrast however, caffeine, which is a major bioactive constituent of coffee, did not show any inhibition at doses of up to 100 μM. Coffee was also found to inhibit UDP-glucuronosyl transferase (UGT) activity towards 1-naphthol in vitro to a similar extent [IC50 = 1.5% (v/v)] as in intact cells. Moreover, the expression of the UGT 1A6, which is known to mainly catalyze the glucuronidation of 1-naphthol, was not affected by coffee. If coffee inhibits placental glucuronidation during pregnancy, its consumption would increase the fetal plasma concentrations of harmful xenobiotics, potentially affecting normal fetal growth and development.

We have identified four cytosolic sulfotransferase (SULT) homologs in the genome database of Drosophila melanogaster, and have designated these genes dmST1-4. Each of these four isozymes was subsequently classified into a different and novel gene family, as determined by the low amino acid sequence homology (less than 40%) between them, and also toward their vertebrate homologs. The transcripts for these four SULT homologs were detectable at all developmental stages in D. melanogaster. In addition, three of these isozymes, the exception being dmST2, were successfully expressed and purified in Escherichia coli. These recombinant dmST1, 3, and 4 products showed high sulfating activity toward phenolic compounds such as vanillin and 4-nitrophenol, but showed no activity toward typical endogenous substrates such as tyramine and serotonin. DmST4 and dmST3 showed the lowest and highest Km values for 3′-phosphoadenosine-5′-phosphosulfate (PAPS) respectively. DmST4 also showed low but not negligible activity toward 20-hydroxyecdysone.

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

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

© 2016 Taylor & Francis. Recent evidence indicates that hypoxia-inducible vascular endothelial growth factor (VEGF) has neurotrophic and neuroprotective effects on neuronal and glial cells. On the other hand, recent epidemiological studies showed that daily coffee consumption has been associated with a lower risk of several neuronal disorders. Therefore, we investigated the effect of coffee on VEGF expression in human neuroblastoma SH-SY5Y cells. We found that even low concentration of coffee (<2%) strongly induced VEGF expression via an activation of HIF-1α. The activation of HIF-1α by coffee was attributed to the coffee-dependent inhibition of prolyl hydroxylation of HIF1α, which is essential for proteolytic degradation of HIF-1α. However, no inhibition was observed at the catalytic activity in vitro. Coffee component(s) responsible for the activation of HIF-1α was not major constituents such as caffeine, caffeic acid, chlorogenic acid, and trigonelline, but was found to emerge during roasting process. The active component(s) was extractable with ethyl acetate. Our results suggest that daily consumption of coffee may induce VEGF expression in neuronal cells. This might be related to protective effect of coffee on neural disorders such as Alzheimer's disease and Parkinson's disease.

© 2014 The Pharmaceutical Society of Japan. Recent epidemiological studies showed that coffee consumption is associated with a lower risk of type 2 diabetes, presumably due to suppression of excess fat accumulation in adipocytes. However, the mechanism underlying the effect of coffee on adipocyte differentiation has not been well documented. To elucidate the mechanism, we investigated the effect of coffee on the differentiation of mouse preadipocyte 3T3-L1 cells. Coffee reduced the accumulation of lipids during adipocytic differentiation of 3T3-L1 cells. At 5% coffee, the accumulation of lipids decreased to half that of the control. Coffee also inhibited the expression of the peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor controlling the differentiation of adipocytes. Furthermore, coffee reduced the expression of other differentiation marker genes, aP2, adiponectin, CCAAT-enhancer-binding protein α (C/EBPα), glucose transporter 4 (GLUT4), and lipoprotein lipase (LPL), during adipocyte differentiation. Major bioactive constituents in coffee extracts, such as caffeine, trigonelline, chlorogenic acid, and caffeic acid, showed no effect on PPARγ gene expression. The inhibitory activity was produced by the roasting of the coffee beans.

Coffee is the beverage consumed during pregnancy that is most commonly associated with adversely affecting the developing fetoplacental unit. Since placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD-2) is an important determinant for maintenance of fetal glucocorticoid levels during pregnancy, we investigated the effect of coffee on 11β-HSD-2 expression in the human placental choriocar-cinoma cell line, JEG-3. Addition of coffee to the culture medium of JEG-3 cells increased 11β-HSD-2 gene expression after 24 hr in a dose-dependent manner. Cellular 11β-HSD-2 activity, which was calculated by the amount of cortisol that was converted to cortisone in the culture medium, also increased in the coffee-treated JEG-3 cells. None of the major constituents of coffee, such as caffeine, caffeic acid or chlorogenic acid, showed any ability to induce 11β-HSD-2 gene expression. The 11β-HSD-2-inducing activity in coffee was extractable with ethyl acetate. Increasing the degree of roasting improved the ability of the coffee extract to induce 11β-HSD-2 gene expression indicating that the active constituent(s) is produced during the roasting process. © 2011 The Pharmaceutical Society of Japan.

The ST-20 and ST-40 cDNAs encode rat liver hydroxysteroid sulfotransferases (HS-ST) that are 90% identical in amino acid sequence but exhibit different substrate preferences for dehydroepiandrosterone (DHEA), androsterone (AD), and cortisol (CS), ST-40 is active for all three substrates, whereas ST-20 is mainly active for cortisol. To determine the domain responsible for the substrate preferences of the HS-STs, 20 chimeric HS-STs were constructed by reciprocal exchanges of DNA fragments derived from the cDNAs and were expressed in Escherichia coli. Some chimeric enzymes were enzymatically active for all three substrates, and some displayed reduced or lost CS-ST activity, with retention of DHEA- and AD-ST activities. Others lost all HS-ST activity. Analysis revealed that a central region (region III spanning amino acids 102164 with five amino acid differences between ST-20 and ST-40) is essential for HS-ST activity, whereas regions II (amino acids 65-101) and IV (amino acids 165219) are unimportant with regard to substrate preference. It was also shown that the parental combination of regions I (amino acids 1-64) and V (amino acids 220-284) is essential for CS-ST activity. Photoaffinity labeling with [35S]3'-phosphoadenosine 5'- phosphosulfate (PAPS) revealed that some inactive chimeras lost affinity for PAPS. These results suggested that an ordered structure formed by regions I, III, and V is required for HS-ST activity, especially for substrate preference and PAPS binding.

We have demonstrated the neuronal localization of neurosteroidogenic systems as well as the regulation of neurosteroidogenic activity in an adult rat hippocampus. The expressions of steroidogenic enzymes (cytochrome P450scc, adrenodoxin (ADX), NADPH-adrenodoxin reductase (ADR), hydroxysteroid sulfotransferase and steroidogenic acute regulatory protein (StAR)) were observed principally in pyramidal neurons and granule neurons by means of immunohistochemical staining. The expression of P450scc, redox partners, sulfotransferase and StAR was also confirmed by Western blot analysis.Exposing the neurons to N-methyl-D-aspartate (NMDA) stimulated the process of active neurosteroidogenesis. Upon stimulation with NMDA, the significant net production of pregnenolone (PREG) and pregnenolone sulfate (PREGS) was observed in the hippocampus. This neurosteroid production was considerably suppressed by the addition of antagonists of NMDA receptors, by Ca2+ depletion or by the addition of an inhibitor of P450scc. Upon stimulation with NMDA, the processing of full-length StAR (37-kDa) to the truncated 30-kDa StAR was observed. Taken together, these observations imply that hippocampal neurons synthesize neurosteroids. This synthesis may be stimulated and regulated by glutamate-mediated synaptic communication. We also discuss the possibility that PREGS may act as a paracrine modulator of NMDA receptors. © 2002 Elsevier Science B.V.

The three-dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method: the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase 1 structure, which suggested that SMase adopts a structure similar to that of DNase I. The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center; are conserved in SMase. In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues. Asp 251 and His 252. A sequence database search showed that the motif is highly specific to mammalian DNase I and bacterial SMase. The functional roles of SMase residues identified by the sequence comparison were consistent with the results from mutant studies. Two Bacillus cereus SMase mutants (H134A and H252A) were constructed by site-directed mutagenesis. They completely abolished their catalytic activity. A model for the SMase-sphingomyelin complex structure was built to investigate how the SMase specifically recognizes its substrate. The model suggested that a set of residues conserved among bacterial SMases, including Trp 28 and Phe 55, might be important in the substrate recognition. The predicted structural similarity and the conservation of the functionally important residues strongly suggest a distant evolutionary relationship between bacterial SMase and mammalian DNase I. These two phosphodiesterases must have acquired the specificity for different substrates in the course of evolution.

Inhibition of mammalian DNA topoisomerase I by phospholipids was investigated using purified enzyme. Acidic phospholipids inhibited the DNA relaxation activity of topoisomerase I whereas neutral phospholipid, phosphatidylethanolamine, did not. Accumulation of a protein-DNA cleavable complex, an intermediate which is known to accumulate upon inhibition by a specific inhibitor camptothecin, did not occur. The filter binding assay revealed that the DNA binding activity of the enzyme was inhibited by acidic phospholipids. Moreoever, direct binding of phosphatidylglycerol to topoisomerase I was demonstrated. These results indicated that the inhibitory effect of acidic phospholipids on topoisomerase I was due to the loss of the DNA binding of the enzyme as a result of direct interaction between phospholipids and the enzyme. © 1990.