Regulation of Iron-Ion Transporter SLC11A2 by Three Identical miRNAs.

Here, we searched for microRNAs (miRNAs) in silico that could interact with SLC11A2 mRNA, a solute carrier (SLC) iron-ion transporter, and investigated their effects on SLC11A2 gene expression using the cultured human colon carcinoma cell line, Caco-2. In silico analysis using the miRWalk2.0 database revealed that several types of miRNAs interact with the human SLC11A2 gene; we focused on three miRNAs, miR-149-5p, miR-362-5p, and miR-539-5p as candidates in this study. We first revealed that the three miRNAs interact with the SLC11A2 3'-untranslated region (3'-UTR) using a luciferase assay in a Caco-2 cell line. We then examined whether the expression of each miRNA affected the expression of SLC11A2 mRNAs and their transcribed transporter proteins. We found transiently expressed miRNAs significantly reduced the reporter activity of the SLC11A2 3'-UTR site in Caco-2 cells by significantly decreasing the SLC11A2 gene and protein expression in the miRNA-transfected Caco-2 cells. Subsequently, we investigated the effects of these miRNAs on SLC11A2's iron-ion transporting activity by measuring iron-ion concentration in Caco-2 cells. Administration of ammonium iron (II) sulfate hexahydrate to Caco-2 cells significantly increased the intracellular iron-ion concentration. However, in iron-ion-pretreated cells, overexpression of each of the three miRNAs resulted in decreased intracellular iron-ion concentration. This indicated that overexpressed miRNAs inhibited iron-ion influx into Caco-2 cells by attenuating SLC11A2 transporting activity. Using in silico analysis, we predicted that three studied miRNAs could bind to the iron-ion influx transporter SLC11A2 and revealed that they regulate SLC11A2 gene expression and iron-ion transporting function in an in vitro system.

Therapeutic Response to Paroxetine in Major Depressive Disorder Predicted by DNA Methylation.

BACKGROUND: Antidepressants have variable therapeutic effects, depending on genetic and environmental factors. Approximately 30% of major depressive disorder (MDD) patients do not respond significantly to antidepressants such as paroxetine, a selective serotonin reuptake inhibitor (SSRI). However, the biological mechanisms behind this phenomenon are mostly unknown. Here, we examined the role of patients' epigenetic background in SSRI efficacy. METHODS: Genome-wide DNA methylation analysis of the peripheral blood of Japanese MDD patients was performed by using the Infinium HumanMethylation450 BeadChip. RESULTS: We compared the results of the 10 patients who best responded to paroxetine (BR) with the 10 worst responders (WR), and found 623 CpG sites with a >10% difference in DNA methylation level. Among them, 218 sites were nominally significant between BR and WR (p < 0.05), and 2 sites (cg00594917 and cg07260927) were significantly different after false discovery rate (FDR) correction (q < 0.05). The methylation difference was greatest at cg00594917, located in the first exon of the PPFIA4 gene, which codes for liprin-α (p = 0.00012). Hierarchical cluster analysis of 23 CpG sites in the PPFIA4 gene distinguished BR and WR, except for 1 WR patient. The cg07260927 site was located in the 5'UTR of the heparin sulfate-glucosamine 3-sulfotransferase 1 (HS3ST1) gene (p = 0.00013). Hierarchical cluster analysis of 28 CpG sites in HS3ST1 distinguished BR and WR, except for 1 WR and 2 BR patients. CONCLUSION: Our results suggest that patients' DNA methylation profile at specific genes such as PPFIA4 and HS3ST1 is associated with individual variations in therapeutic responses to paroxetine.

Identification of Candidate Target Cyp Genes for microRNAs Whose Expression Is Altered by PCN and TCPOBOP, Representative Ligands of PXR and CAR.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in mRNA post-transcriptional regulation. The deregulation of miRNAs affects the expression of drug-metabolizing enzymes, drug transporters, and nuclear receptors, all of which are important in regulating drug metabolism. miRNA expression can be altered by several endogenous or exogenous agents, such as steroid hormones, carcinogens, and therapeutic drugs. However, it is unclear whether hepatic miRNA expression is regulated by nuclear receptors, such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR), which are indispensable for the expression of the CYPs. Here we investigated the effects of the mouse PXR and CAR ligands pregnenolone-16α-carbonitrile (PCN) and 1,4-bis[(3,5-dichloropyridin-2-yl)oxy]benzene (TCPOBOP) on hepatic miRNA expression in mice. We found that the expression of 9 miRNAs was increased (>2-fold) and of 4 miRNAs was decreased (>50%) in response to PCN, while TCPOBOP treatment led to the up-regulation of 8 miRNAs and down-regulation of 6 miRNAs. Using several miRNA target prediction algorithms, we found that the predicted target genes included several lesser known Cyp genes (Cyp1a1, Cyp1b1, Cyp2b10, Cyp2c38, Cyp2u1, Cyp4a12a/b, Cyp4v3, Cyp17a1, Cyp39a1, and Cyp51). We analyzed the expression of these genes in response to PCN and TCPOBOP and found changes in their mRNA levels, some of which were negatively correlated with the expression of their corresponding miRNAs, suggesting that miRNAs may play a role in regulating Cyp enzyme expression. Further studies will be required to fully elucidate the miRNA regulatory mechanisms that contribute to modulating CYP expression.

Luteolin attenuates doxorubicin-induced cytotoxicity to MCF-7 human breast cancer cells.

Luteolin, a flavone found in some vegetables, has been reported to exhibit antioxidant, antiinflammatory, and anticancer activities. In the present study, we found that luteolin has biphasic effects on the viability of the human breast cancer cell line MCF-7. That is, cell viability increased at relatively low luteolin concentrations and decreased at relatively high concentrations. Focusing on the proliferative effect at low concentrations, we showed that luteolin has a cytoprotective effect on MCF-7 cells when administered with doxorubicin. Moreover, luteolin attenuated doxorubicin-induced cytotoxicity even in the presence of the estrogen receptor (ER) antagonist ICI 182,780 and the ER-negative MDA-MB-453 human breast cancer cell line. Reactive oxygen species (ROS) were generated after doxorubicin treatment of MCF-7 cells. In contrast, luteolin attenuated doxorubicin-induced ROS generation. Levels of the antiapoptotic protein Bcl-2 in luteolin-treated MCF-7 cells were significantly higher than those in doxorubicin-treated MCF-7 cells. Our results suggest that a low concentration of luteolin attenuates doxorubicin-induced cytotoxicity to MCF-7 cells through a combination of antioxidant activity and an increase in levels of Bcl-2 protein.

Effect of lipopolysaccharide on the xenobiotic-induced expression and activity of hepatic cytochrome P450 in mice.

Infection-associated inflammation can alter the expression levels and functions of cytochrome P450s (CYPs). Cyp gene expression is regulated by the activation of several nuclear receptors, including pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR). These receptors can be activated by xenobiotics, including medicines. Here, to study the xenobiotic-induced fluctuations in CYP during inflammation, we examined the effect of lipopolysaccharide (LPS) treatment on the level of mRNAs encoding hepatic CYPs induced by xenobiotic-activated nuclear receptors, in mice. Both the mRNA induction of Cyp genes and the metabolic activities of CYP proteins were examined. LPS treatment caused a significant decrease in the induced expression of the mRNAs for Cyp3a11, 2c29, 2c55, and 1a2, but not for Cyp2b10. To assess the CYP enzymatic activities, CYP3A-mediated testosterone 6ß-hydroxylation and the intrinsic clearance (CL(int)) of nifedipine in liver microsomes were measured in mice treated with the xenobiotic pregnenolone-16alpha-carbonitrile (PCN) with or without LPS administration. Both assays revealed that the CYP3A activity, which was induced by PCN, declined significantly after LPS treatment, and this decline correlated with the Cyp3a11 mRNA level. In addition, we found that the mRNAs for interleukin (IL)-1ß and tumor necrosis factor (TNF) α were increased after treatment with LPS plus xenobiotics. Our findings demonstrated that LPS treatment reduces the PXR- and AhR-mediated, and possibly CAR-mediated Cyp gene expression and further suggest that these decreases are dependent on inflammatory cytokines in the liver.

Involvement of an influx transporter in the blood-brain barrier transport of naloxone.

Naloxone, a potent and specific opioid antagonist, has been shown in previous studies to have an influx clearance across the rat blood-brain barrier (BBB) two times greater than the efflux clearance. The purpose of the present study was to characterize the influx transport of naloxone across the rat BBB using the brain uptake index (BUI) method. The initial uptake rate of [(3)H]naloxone exhibited saturability in a concentration-dependent manner (concentration range 0.5 microM to 15 mM) in the presence of unlabeled naloxone. These results indicate that both passive diffusion and a carrier-mediated transport mechanism are operating. The in vivo kinetic parameters were estimated as follows: the Michaelis constant, K(t), was 2.99+/-0.71 mM; the maximum uptake rate, J(max), was 0.477+/-0.083 micromol/min/g brain; and the nonsaturable first-order rate constant, K(d), was 0.160+/-0.044 ml/min/g brain. The uptake of [(3)H]naloxone by the rat brain increased as the pH of the injected solution was increased from 5.5 to 8.5 and was strongly inhibited by cationic H(1)-antagonists such as pyrilamine and diphenhydramine and cationic drugs such as lidocaine and propranolol. In contrast, the BBB transport of [(3)H]naloxone was not affected by any typical substrates for organic cation transport systems such as tetraethylammonium, ergothioneine or L-carnitine or substrates for organic anion transport systems such as p-aminohippuric acid, benzylpenicillin or pravastatin. The present results suggest that a pH-dependent and saturable influx transport system that is a selective transporter for cationic H(1)-antagonists is involved in the BBB transport of naloxone in the rat.

Apoptosis-like cell death of human breast cancer cell line MCF-7 induced by buprenorphine hydrochloride.

The analgesic buprenorphine hydrochloride (Bph) induced apoptosis-like cell death in the caspase-3-deficient human breast cancer cell line, MCF-7. This apoptosis-like cell death activated key molecules in the mitochondrial apoptotic pathway: cytochrome c, caspase-9, caspase-7, and caspase-6. Bph caused the release of fluorescent protein from the mitochondria of MCF-7 cells transfected with the pDsRed2-Mito-vector in a time-dependent manner, suggesting disruption of the mitochondrial membrane. Zn(2+) as high as 2 mM did not inhibit the DNase that took part in this apoptosis. Thus, this unidentified DNase might resemble other DNases involved in apoptosis-like cell death whose activity is not inhibited by zinc ion.

Whole-body X-irradiation induces acute and transient expression of heme oxygenase-1 in rat liver.

Activation of the stress-inducible heme oxygenase-1 (HO-1) gene by X-irradiation was investigated in rat liver. When male Wistar MS strain rats (8 weeks) received whole-body irradiation of 17.0 Gy, 7 h later the activity of heme oxygenase in the liver was significantly enhanced (2.5 times). The level of HO-1 mRNA expression was increased by 2.3 and 4.0 times 2 and 4 h after radiation, and then declined at 7 and 10 h to the level of 2.0 and 1.6 times of the control. When the X-ray dose was varied from 4.0 to 21.7 Gy, the transcription of the gene was enhanced at all doses and the level of activation was dose-dependent. Finally, western blotting of irradiated liver demonstrated a significant increase in the level of HO-1 induced by X-rays, peaking at 4 h. Thus, X-rays were confirmed to be stressors that induce acute HO-1 expression transiently in the liver.

Expression of the polyubiquitin gene early in the buprenorphine hydrochloride-induced apoptosis of NG108-15 cells.

To clone genes expressed early in the buprenorphine hydrochloride (Bph)-induced apoptosis of NG108-15 nerve cells, we adopted a previously reported rapid and simple differential display (DD) cloning procedure. Complementary DNA was generated from differentially expressed mRNAs by reverse transcription (RT) using a fully degenerate 6-mer oligonucleotide as the primer. PCR amplification was then conducted using a combination of three arbitrary but defined 10-bp nucleotide primers. The differentially generated DNA fragments were detected by agarose gel electrophoresis, and 9 were excised from the gel and subcloned into a sequencing vector. Three DNAs that were specifically expressed upon Bph-induced apoptosis of NG108-15 cells were sequenced. Their specific expression was then confirmed by reverse-transcription PCR. One was identified as the mouse polyubiquitin gene c, and the others remain unidentified. Northern and western blots indicated the transcription and translation of polyubiquitin early in Bph-induced apoptosis. The polyubiquitination of apoptotic cellular proteins was also confirmed.

Genomic cloning of Xenopus TFIIS (TCEA1) and identification of its transcription start site.

We previously cloned the cDNA of the transcription factor TFIIS (SII) from Xenopus laevis and showed that its expression was not constant during Xenopus development. To investigate its regulation, we cloned the genomic DNA of Xenopus TFIIS, focusing on the 5'-promoter region. Here, we present the Xenopus TFIIS genomic sequence (-1730 to +214) and transcription start site (cap site). We define the position of the primary cap site as the adenine located 142 bp upstream from the adenine of the ATG (Met) codon. Another putative start-site region, where 13 transcriptional start sites are clustered within 12 bp, was mapped about 100 bp downstream of the primary cap site. Although a computer search found putative trans-element binding sites proximal to two Xenopus TFIIS transcription start sites, we could not identify typical "TATA" or "CAATT" boxes upstream of the primary cap site, probably owing to TFIIS's character as a "house keeping gene".

[Effect of bcl-2 over-expression on the membrane structure of NG108-15 nerve cells: effectiveness of membrane-structure analysis using FACS for the first screening of apoptosis-inducing drugs].

Bcl-2 is a Bcl-2 family protein that is known to be anti-apoptotic and is predominantly localized to the mitochondria. We previously showed that an analgesic, buprenorphine hydrochloride (Bph), induces apoptosis in the rodent-derived nerve cell line, NG108-15, through the mitochondrial apoptotic route. A Bcl-2-overexpressing strain of NG108-15 cells, Bcl-2 (P2), was established, and the effect of Bcl-2 expression on Bph-induced apoptosis was compared between the mock vector-transfected NG108-15 cells and the Bcl-2 (P2) cells. The Bcl-2 (P2) cells died after treatment with Bph, and we observed all the biological and morphological markers of apoptosis that we tested for. In flow cytometric analysis, a difference in the cell membrane phospholipid flip-flop pattern-a feature of apoptosis- was observed between the NG108-15 cells and the Bcl-2 (P2) cells. Here, we show by flow cytometric analysis that Bcl-2 over-expression may affect the membrane structure of Bcl-2 (P2) cells. An increased fluorescein isothiocyanate (FITC) signal of annexin V-FITC, which typically represents phospholipid flip-flop of the cellular membrane in early apoptosis, was barely detected in the Bcl-2 (P2) cells. Since our previous study reported the localization of over-expressed Bcl-2 protein to the cell membrane of Bcl-2 (P2) cells, together these observations suggest that the Bcl-2 protein may affect the integrity of the structure of the NG108-15 cell membrane.

Blood-brain barrier transport of naloxone does not involve P-glycoprotein-mediated efflux.

The blood-brain barrier (BBB) transport of naloxone, a potent and specific opioid antagonist, was investigated in rats using the brain uptake index method and the brain efflux index method. The apparent influx clearance of [(3)H]naloxone across the BBB was 0.305 mL/min/g brain. [(3)H]naloxone was eliminated from the brain with an apparent elimination half-life of 15.1 min after microinjection into the parietal cortex area 2 regions of the rat brain. The apparent efflux clearance of [(3)H]naloxone across the BBB was 0.152 mL/min/g brain, which was calculated from the elimination rate constant (4.79 x 10(-2) min(-1)) and the distribution volume in the brain (3.18 mL/g brain). The influx clearance across the BBB was two times greater than the efflux clearance. The elimination of [(3)H]naloxone from the brain was not inhibited in the presence of the typical P-glycoprotein (P-gp) inhibitors such as quinidine, verapamil, vinblastine, and vincristine, indicating that naloxone is not a P-gp substrate in the rat. In vitro experiments by using human multidrug resistance 1 (MDR1)/P-gp overexpressing HeLa cells showed that the uptake of naloxone by the cells did not change in the presence of the P-gp inhibitors. In conclusion, the present results obtained from in vivo and in vitro studies suggest that P-gp is not involved in the BBB transport of naloxone.

Evaluation of cell death caused by CDF (cyclophosphamide, doxorubicin, 5-fluorouracil) multi-drug administration in the human breast cancer cell line MCF-7.

We evaluated the features of cell death induced by CDF (cyclophosphamide [CPA], doxorubicin [DOX], 5-fluorouracil [5-FU]) multi-drug administration in vitro using the human breast cancer cell line MCF-7. Used individually, DOX and 5-FU induced 60% cell death in MCF-7 cells, at 5 microg/ml and 25 microg/ml, respectively, by the 4th day following drug treatment. CPA was the least cytotoxic of the 3 drugs, causing only 20% cell death, even at the high concentration of 500 microg/ml. Treating cells with a mixture of all three anticancer drugs resulted in 60% cell death, on the second and third day following drug treatment. The nature of the cytotoxicity of CPA, DOX, and 5-FU was investigated, because these drugs are sometimes used to induce apoptosis. Biochemical analysis showed faint DNA fragmentation in the case of DOX or all three drugs, but not for treatment with CPA or 5-FU. In contrast, the morphological apoptotic feature of a condensed nucleus was observed only for CPA and 5-FU. Flow cytometric data agreed with the morphological results in that the FACS cytogram for DOX and for all three drugs was different from that for CPA or 5-FU given alone. These observations suggested that the cell death induced by these anticancer drugs in the human breast cancer cell line MCF-7 is a mixture of apoptotic and non-apoptotic, but it becomes completely non-apoptotic in the case of multi-drug administration.

Over-expressed Bcl-2 cannot suppress apoptosis via the mitochondria in buprenorphine hydrochloride-treated NG108-15 cells.

We previously reported that the morphine alkaloid derivative buprenorphine hydrochloride (Bph) induces rapid apoptosis in NG108-15 nerve cells accompanied by the activation of caspase-3. Here, we found this kind of apoptosis was also accompanied by rapid loss of the mitochondrial membrane potential, followed by the efflux of cytochrome c from the mitochondria to the cytosol and the activation of caspases-9 and -3. Together, these results strongly suggested the Bph death signal was routed through the mitochondrial pathway in NG108-15 cells. In these cells, serum-starvation induces a different apoptosis, which we exploited to investigate Bcl-2's role as an apoptosis inhibitor. We made an NG108-15 transfectant, Bcl-2(P2), that stably expressed human Bcl-2, and used it to test Bcl-2's effect on the serum-starvation-induced apoptosis in NG108-15 cells. Cell viability, DNA-ladder formation, and efflux of cytochrome c from the mitochondria were all detected, showing that the human Bcl-2 functioned normally in the Bcl-2(P2) cells. Although the apoptotic events tested were identical in the parental cells and transformants, Bcl-2 expression completely failed to inhibit Bph-induced apoptosis in the Bcl-2(P2) cells.

Expression of transcriptional repressor NC2alpha during Xenopus early embryogenesis.

To study the mechanisms behind the suppression of gene expression in the early phase of Xenopus development (cleavage stage), we conducted in silico cloning of the Xenopus transcriptional repressor NC2alpha. A search of the GenBank EST database using human NC2alpha as a probe identified Xenopus mitotic phosphoprotein 30 (xMP30) as a prime candidate for Xenopus NC2alpha (xNC2alpha). Full-length cDNA sequencing showed that xNC2alpha/xMP30 had a 68.9% identity at the amino acid level with its human counterpart. Northern blotting showed that xNC2alpha existed abundantly as a maternal mRNA. After the fertilization, the expression of xNC2alpha rapidly increased and reached a maximum 3 h before midblastula transition (MBT). Then its expression gradually decreased toward the early neurula stage. The expression profile of xNC2alpha mRNA is compatible with that of xNC2beta, which is the other component of the Xenopus NC2 transcriptional repressor.