The tumor microenvironment is now recognized as a major factor that influences not only the response to conventional anti-cancer therapies but also helps define the potential for malignant progression and metastasis. In particular, hypoxia is now considered a fundamentally important characteristic of the tumor microenvironment. Furthermore, discovery of the hypoxia inducible factor 1α (HIF-1α) has led to a rapidly increasing understanding of the molecular mechanisms involved in tumor hypoxia. This in turn has led to the current extensive interest in the signal molecules related to tumor hypoxia as potential molecular targets for cancer therapeutics. In this paper we give an overview of recent advances in hypoxia research, including cancer treatments that target tumor hypoxia. Progress in the development of hypoxia-targeting drugs will be discussed, including antiangiogenic hypoxic cell radiosensitizers and hypoxic cytotoxins, hypoxia targeting boron carriers and p53-inhibiting bifunctional radiosensitizers. We will also review our own recent research results in these areas. For example, we have found that certain of the 2-nitroimidazole radiosensitizers and heterocycle-N-oxide hypoxic cytotoxins we developed have antiangiogenic activity and antimetastatic activity. We propose that these activities are based on the inhibition of signal transduction mediated by HIF-1α. The anti-tumor activities of hypoxia response are considered to be cytostatic (tumor dormancy-inducing) effects in contrast to cytotoxic DNA damaging effects. The combinaiton of these cytostatic effects that are related to radiosensitization with the cytotoxic effects of radiation should improve the prognosis and QOL of patients receiving radiation and lead to an overall response to treatment. Based on these considerations, we developed the antiangiogenic hypoxic cell radiosensitizers, TX-1877, TX-1898 and the hypoxic cytotoxin TX-402 that inhibits the HIF-1α pathway We will also discuss our research involved with the development of other drugs to exploit tumor hypoxia, including a hypoxia-targeting boron carrier for boron neutron capture therapy (BNCT) and a p53 inhibiting radiosensitizer.
Lymphocyte homing is mediated by a specific interaction between the lymphocyte homing receptor L-selectin and its sulfated glycoprotein ligands, which are expressed on high endothelial venules (HEV) in the lymph nodes. To examine the significance of the sulfation of L-selectin ligands, our group has generated gene-targeted mice deficient in both N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2. The mutant mice show approximately 75% less lymphocyte homing to the peripheral lymph nodes than normal, indicating that GlcNAc6ST-1 and GlcNAc6ST-2 play a major role in the biosynthesis of L-selectin ligand in HEV. In agreement with this interpretation, an oligosaccharide analysis indicated that 6-sulfo sialyl Lewis X, a major L-selectin ligand sulfated glycan, is almost completely abrogated in the double-deficient mice. Lymphocyte homing into the parenchyma of lymph nodes is mediated by a series of interactions: rolling, activation by chemokines, integrin-mediated adhesion, and transmigration. During the rolling interaction, which is mediated by L-selectin and sulfated glycans, lymphocytes receive activation signals from chemokines presented on the surface of HEV by heparan sulfate, a sulfated glycosaminoglycan, which leads to the activation of lymphocyte β2 integrin. Sulfated glycans are thus involved in both the rolling and the chemokine-induced activation steps between lymphocytes and HEV. In this article, recent findings on the roles of sulfated glycans in both of these lymphocyte-homing steps will be reviewed. The possible application of sulfated glycans for the prevention of inflammatory disorders will also be discussed.
The feasibility of transgenic tobacco, engineered to express bacterial polyphosphate (polyP), for phytoremediation of mercury pollution was evaluated. T3 progeny of the transgenic tobacco produced a large amount of polyP in leaves and showed a relatively high resistant phenotype to Hg2+ than its wild-type progenitors. These results suggest that the integrated ppk gene, encoding polyphosphate kinase (PPK), a key enzyme for polyP biosynthesis, is stably conserved in tobacco genome, and translated to active PPK which catalyzed biosynthesis of polyP, and suggest that polyP is capable of reducing the cytotoxicity of Hg2+, probably via chelation formation with polyP. The transgenic tobacco expressing polyP accumulated significantly more mercury than its wild-type progenitors from Hg2+-containing agar medium and simulated soils without taxing the tobacco plants suggesting that the transported Hg2+ was accumulated as a less toxic Hg-polyP complex in the tobacco tissues. Based on the results obtained in the present study, the polyP-mediated accumulation of mercury from mercurial-contaminated soils may provide an ecologically compatible approach for phytoremediation of mercury pollution.
The purpose of this study was to determine the level of 4-(4-bromophenyl)-4-hydroxypiperidine (BPHP), a bromperidol (BRO) metabolite, in rat plasma by HPLC with fluorescence detection after pre-column derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). After basic extraction of the samples with benzene, derivatization with NBD-F was conducted in borate buffer (pH 8.0) at 60 °C for 3 min. Mexiletine was utilized through the procedure as an internal standard (IS). Retention times of the BPHP and IS derivatives were 7.7 and 11.5 min, respectively. The regression equation for BPHP showed good linearity in the range of 0.01—1 mg/ml with the detection limit of 0.003 μg/ml. The coefficient of variation was less than 12.0%. The recovery was satisfactory. This method was applied for a pharmacokinetic study of BPHP in comparison with 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), the corresponding haloperidol (HAL) metabolite, in rats. The ratio of the area under the plasma concentration curve (AUC) after p.o. administration of BPHP to the AUC after i.p. administration of BPHP (46%) was lower than that of CPHP (56%), indicating that intestinal absorption of BPHP is lower than that of CPHP. The ratio of BRO metabolism to BPHP (48%) was 1.8-fold higher than that of HAL metabolism to CPHP (27%); the ratio was estimated as (AUCp.o.,A→B/AUCp.o.,B)×100, where AUCp.o.,A→B is the AUC value of BPHP or CPHP after p.o. administration of BRO or HAL, and AUCp.o.,B is the AUC of BPHP or CPHP after administration of BPHP or CPHP, respectively. Our method provides a sensitive procedure for determination of BPHP in rat plasma and is suitable for pharmacokinetic studies of BPHP after BRO administration.
The in vitro metabolism of (+)-fenchone was examined in human liver microsomes and recombinant enzymes. Biotransformation of (+)-fenchone was investigated by gas chromatography-mass spectrometry. (+)-Fenchone was found to be oxidized to 6-exo-hydroxyfenchone, 6-endo-hydroxyfenchone and 10-hydroxyfenchone by human liver microsomal P450 enzymes. The formation of metabolite of (+)-fenchone was determined by relative abundance of mass fragments and retention time with GC. CYP2A6 and CYP2B6 in human liver microsomes were major enzymes involved in the hydroxylation of (+)-fenchone, based on the following lines of evidence. First, of eleven recombinant human P450 enzymes tested, CYP2A6 and CYP2B6 catalyzed oxidation of (+)-fenchone. Second, oxidation of (+)-fenchone was inhibited by thioTEPA, (+)-menthofuran anti-CYP2A6 and anti-CYP2B6 antibodies. Finally, there was a good correlation between CYP2A6, CYP2B6 contents and (+)-fenchone hydroxylation activities in liver microsomes of 8 human samples.
This study investigated the protective effects of a group IIA secretory phospholipase A2 (sPLA2-IIA) inhibitor, ochnaflavone, on the progression of carbon tetrachloride (CCl4)-induced acute liver injury in rat liver microsomes in vitro. When rat liver was incubated at 37 °C in the presence of CCl4, the level of phosphatidylethanolamine (PE) degradation increased markedly compared with the control. The rat 14 kDa platelet PLA2 antibody, R377, suppressed the degradation of PE. Pretreating the microsome with ochnaflavone (2—16 μM) reduced the level of PE degradation in a dose dependent manner. In addition, p-bromophenacy bromide (p-BPB), which is a PLA2 inhibitor, also inhibited PE degradation. However, the inhibitory activity was weaker than that of ochnaflavone. Further investigation showed that ochnaflavone not only inhibited the purified rat platelet sPLA2 activity in a dose dependent manner with an IC50 value of 3.45 μM, when arachidonyl PE was used as a substrate, but also inhibited lipid peroxidation in a dose dependent manner with an IC50 value of 7.16 μM. This result suggests that ochnaflavone prevents the progression of CCl4-induced PE hydrolysis by inhibiting the endogenous sPLA2 activity.
The substrate selectivity of monoamine oxidase A (MAO-A), monoamine oxidase B (MAO-B), diamine oxidase (DAO), and semicarbazide-sensitive amine oxidase (SSAO) was investigated in the absence of chemical inhibitors using the COS-1 cells expressed with respective amine oxidase. Serotonin (5-hydroxytryptamine), 1-methylhistamine, and histamine were preferentially oxidized by MAO-A, SSAO, and DAO, respectively, at a low substrate concentration. In contrast, benzylamine, tyramine, and β-phenylethylamine served as substrates for all of MAO-A, MAO-B, and SSAO. Each amine oxidase showed broad substrate selectivity at a high substrate concentration. The cross-inhibition was remarkable in MAO-A and MAO-B, especially in MAO-A, but not in SSAO and DAO. A study of the substrate selectivity of amine oxidases should include consideration of the effects of substrate concentration and specific chemical inhibitors.
The loss of organelles and DNA is important to ensure transparency of the lenses, and DNase II-like acid DNase (also called DNase IIβ, DLAD) is related to the loss of organelles and DNA in the lenses. We investigated the relation between the degradation of DNA and DLAD mRNA expression in the lenses of two hereditary cataract rats, the UPL rat (UPLR) and the Shumiya cataract rat (SCR), during cataract development. Undigested DNA was detected in the lens cortexes of normal UPLRs and SCRs, and undigested DNA was degraded in the lens nuclei of normal UPLRs and SCRs. DLAD does not affect common cataract formation, since DLAD mRNA expression levels in the lenses of cataractous SCRs were not changed with an increase in age, and undigested DNA was degraded in the lens nuclei of cataractous SCRs. On the other hand, an accumulation of undigested DNA was found in the lens nuclei of cataractous UPLRs at 46 and 53 d of age with opaque lenses, and the decrease in DLAD mRNA expression levels occurred prior to the accumulation of undigested DNA in the lens nuclei. It is possible that UPLRs are a good model for cataract caused by a decrease of DNA degradation in the lenses.
Regulation of fucosyltransferases (FUTs) and sialyltransferases (STs) in a human colon adenocarcinoma cell line HT-29 and nuclear factor κB (NFκB)-p65 knockdown HT-29 cells was investigated after stimulation with tumor necrosis factor α (TNFα) using real time PCR. TNFα stimulation induced the biphasic increases in expression of NFκB-p65, ST3Gal I, FUT IV, ST3Gal IV and ST6GalNAc III mRNAs and the transient increase in expression of ST6Gal I mRNA and the decrease in ST3GalNAc IV mRNA. In NFκB-p65 knockdown HT-29 cells, the biphasic and transient increases in all of these mRNA expression induced with TNFα were diminished. On the other hand, NFκB-p65 siRNA enhanced the constitutive expression levels of ST3GalNAc IV mRNA which was suppressed by TNFα. Transcription activities of ST3Gal I reporter gene from nt −1050 5′-flanking region to translation initiation site which has consensus NFκB binding sites were up-regulated by stimulation with TNFα in HT-29 cells. The promoter activities for deletion constructs of each NFκB binding sites were determined using dual luciferase assay. The results indicated that constitutive promoter activities were detected at nt −120 5′-flanking translation initiation site and TNFα enhanced ST3Gal I gene expression through NFκB binding sites in HT-29 cells. Combination of stimulation with TNFα and NFκB knockdown with siRNA is useful for determination of NFκB dependent transcriptional regulation.
The present study was carried out to evaluate the anti-inflammatory effect of Spirulina fusiformis on adjuvant-induced arthritis in mice. Arthritis was induced by intra dermal injection of complete freund's adjuvant (0.1 ml) into the right hind paw of Swiss albino mice. Spirulina fusiformis (800 mg/kg/b.wt) was orally administered for 8 d (from 11th to 18th day) to arthritic animals after adjuvant injection. The anti-inflammatory activity of Spirulina fusiformis was assessed by measuring paw volume, body weight, levels of lysosomal enzymes, tissue marker enzymes and glycoproteins in control and experimental animals. In adjuvant-induced arthritic animals, the levels of lysosomal enzymes, tissue marker enzymes, glycoproteins and the paw volume were increased significantly. However the body weight was found to be reduced when compared to control animals. Oral administration of Spirulina fusiformis (800 mg/kg/b.wt) significantly altered these above physical and biochemical changes observed in arthritic animals to near normal conditions. Hence results of this study clearly indicate that Spirulina fusiformis has promising anti-inflammatory activity against adjuvant-induced arthritic animals.
AKR1C12, a mouse member of the aldo-keto reductase (AKR) superfamily, is highly expressed in the stomach and is identical to a protein encoded in an interleukin-3-regulated gene in mouse myeloid cells, but its function remains unknown. In this study, the recombinant AKR1C12 was purified to homogeneity and the specificity for coenzymes and substrates was examined at a physiological pH of 7.4. The enzyme reduced various α-dicarbonyl compounds, several ketosteroids, aldehydes and some ketones using NADH as the preferred coenzyme. In the reverse reaction, the enzyme showed coenzyme preference for NAD+, and oxidized 3α-, 17β- and 20α-hydroxysteroids, and non-steroidal aliphatic and alicyclic alcohols, of which many hydroxysteroids and geranylgeraniol were good substrates, exhibiting low Km and high kcat/Km values. The results, together with the intracellular high ratio of NAD+/NADH, suggest that AKR1C12 functions as a dehydrogenase for the endogenous hydroxysteroids and geranylgeraniol in mouse stomach and myeloid cells.
Four novel peptides, polistes-mastoparan-R1, 2, 3, and polistes-protonectin, were isolated from the venom of a paper wasp, Polistes rothneyi iwatai. MALDI-TOF MS analysis of a small amount of the crude venom gave six molecular-related ion peaks. Among them, m/z 1565 was expected to be a novel peptide. Purification of the crude venom by HPLC gave two known kinins, Thr6-bradykinin and Ala-Arg-Thr6-bradykinin, and four novel peptides named polistes-mastoparan-R1, 2, and 3, and polistes-protonectin. Polistes-mastoparan-R1, 2, and 3 (Pm-R) were tetradecapeptides that possess high sequence homology with that of mastoparan. The sequence of polistes-protonectin was similar to that of protonectin isolated from a Brazilian paper wasp. Histamine-releasing activities of Pm-R1, 2, and 3 were more potent than that of mastoparan. Polistes-protonectin exhibited the most potent hemolytic activity in comparison with the four novel peptides and mastoparan.
A recombinant rat aminopeptidase-B (Ap-B) was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli BL21 harboring a plasmid pGEX-Ap-B and was purified by glutathione-Sepharose 4B and Q-Sepharose columns. The metal-substituted derivatives of Ap-B, Co(II)- and Cu(II)-Ap-B contain almost 1 mole of cobalt(II) and copper(II) ions per enzyme molecule, respectively. The specific activity of Co(II)-Ap-B is very similar to that of recombinant Ap-B but that of Cu(II)-Ap-B is very low. The dissociation constants of the zinc ions of recombinant Ap-B and of the cobalt ions of Co(II)-Ap-B calculated from the relationships between the free metal ions and the residual enzyme activities are 3.7(±1.0)×10−13 and 4.7(±1.0)×10−12 M, respectively. The EPR parameters (g⊥, g// and A//) of Cu(II)-Ap-B were 2.06, 2.27, and 156×10−4 cm−1. The A// value and the g// of Cu(II)-Ap-B are very similar to those of Cu(II)-thermolysin or Cu(II)-dipeptidyl peptidase III, in which the coordination geometry is a distorted tetrahedral.
Two pigmented compounds from Uroleucon nigrotuberculatum, rhododactynaphin-jc-1 (H427) and rhododactynaphin-jc-2 (H373), significantly diminished the cell viability of HL60 cells with IC50 of 10 μM and 30 μM, respectively, in an 18 h-dye uptake assay. Both H427 and H373 augmented the levels of intracellular reactive oxygen species (ROS) and induced apoptosis as demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis. ROS augmentation by both H427 and H373 was inhibited by N-acetylcysteine (NAC) and α-tocopherol. The apoptosis induced by H427 was inhibited efficiently with NAC and caspase-8 inhibitor but less efficiently with α-tocopherol and caspase-9 inhibitor. These findings suggested that these pigments have pro-apoptotic activities via oxidative stress.
San-Zhong-Kui-Jian-Tang (SZKJT; Japanese name: Sanshu-kaigen-to), a traditional Chinese medicine prescription, has been used for treating patients with various cancers. This study first investigates the anticancer effect of SZKJT in two human breast cancer cell lines, MCF-7 and MDA-MB-231. SZKJT exhibited effective cell growth inhibition by inducing cancer cells to undergo G0/G1 phase arrest and apoptosis. Blockade of cell cycle was associated with increased p21/WAF1 levels, and reduced amounts of cyclinD1, cyclinD2 in a p53-independent manner. SZKJT treatment triggered the mitochondrial apoptotic pathway indicated by changing Bax/Bcl-2 ratios, cytochrome c release and caspase-9 activation, but did not act on Fas/Fas ligand pathways and the activation of caspase-8. Further investigation revealed that SZKJT's inhibition of cell growth effect was also evident in a nude mice model. Taken together, our study suggests that the induction of p21/WAF1 and activity of the mitochondrial apoptotic system may participate in the antiproliferative activity of SZKJY in human breast cancer cells.
We have previously reported that INHAT (inhibitor of acetyltransferases) complex subunits, TAF (template activating factor)-Ialpha, TAF-Ibeta and pp32 can inhibit histone acetylation and HAT (histone acetyltransferase)-dependent transcription by binding to histones. Evidences are accumulating that INHAT complex subunits have important regulatory roles in various cellular activities such as replication, transcription, and apoptosis etc. However, how these subunits interact each other remains largely unknown. Using immunoprecipitation (IP) and protein–protein interaction assays with TAF-Ibeta and pp32 deletion mutant proteins, we identify INHAT complex subunits, TAF-Ibeta and pp32 interaction requires highly acidic C-terminal domain of pp32. We also show that the interaction between the INHAT complex subunits is stronger in the presence of histones. In this study, we report that the synergistic inhibition of HAT-mediated transcription by TAF-Ibeta and pp32 is dependent on the highly acidic C-terminal domain of pp32.
Magnolol has been reported to have an inhibitory effect on tumor invasion in vitro and in vivo. In this study, we found that treatment with 30 μM magnolol exhibited growth inhibition partly by inducing apoptosis in cultured human leukemia U937 cells and that the apoptosis was induced via the sequential ordering of molecular events; 1) a transient decrease of phosphorylated extracelluar signal-requlated kinase (ERK), 2) translocation of apoptosis inducing factor (AIF) from mitochondria to cytosol concurrent with a decreased membrane potential, and 3) downregulation of bcl-2 protein. Pretreatment of the cells with a pan-caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) did not prevent the apoptosis induced by magnolol. These findings indicated that the above-mentioned sequence of intracellular signaling events led to apoptosis in magnolol-treated U937 cells, which was caspase-independent.
Icariin is one of the major active flavonoids constituents of Epimedium brevicornum MAXIM (Berberidaceae). Icariin and E. brevicornum have a wide range of pharmacological activities. Abnormality in the hypothalamic-pituitary-adrenal (HPA) axis is considered to be a key neurobilogical factor in major depression, and cytokines have a close relationship with the activation of the HPA axis. In the present study, the aim was to determine whether icariin possesses an antidepressant-like activity, and to explore the effects of icariin on the HPA axis and cytokine levels in chronic mild stress (CMS) model of depression in Sprague-Dawley rats. Icariin significantly increased the sucrose intake of CMS-treated rats from week 3. It not only attenuated the CMS-induced increases in serum corticotropin-releasing factor (CRF) and cortisol levels, but also reversed the abnormal levels of serum interleukin-6 (IL-6) and tumor-necrosis-factor alpha (TNF-α) to the normal in the stressed rats. These results suggested that icariin possessed an antidepressant-like property that was at least in part mediated by neuroendocrine and immune systems.
Indole-3-acetic acid (IAA) activation by horseradish peroxidase (HRP) has been suggested as a new cancer therapy. Interestingly, we found that ultraviolet B (UVB) radiation also can activate IAA and produce free radicals in a dose-dependent manner. In this study, we attempted to identify the free radicals generated by UVB-irradiated IAA (IAAUVB), and to determine whether IAAUVB can induce the apoptosis of G361 human melanoma cells. Since IAA/HRP produces reactive oxygen species (ROS), we examined whether IAAUVB-generated radicals include ROS. Our results show that IAAUVB-induced free radical production is not inhibited by catalase, superoxide dismutase, or sodium formate, indicating that ROS are not generated by IAAUVB. On the other hand, IAAUVB caused lipid peroxidation, and this was blocked by Trolox, a water-soluble vitamin E derivative. Moreover, we found that IAAUVB caused apoptotic cell death and that this was inhibited by a low temperature. We further investigated IAAUVB-mediated apoptotic pathways, and found that IAAUVB causes caspase-8, Bid, caspase-3 activation, and poly (ADP-ribose) polymerase (PARP) cleavage. In addition, these apoptotic pathways were also blocked by low temperature. From these results, we propose that IAAUVB-induced free radicals cause human melanoma cell apoptosis via a death receptor-mediated apoptotic pathway.
9α-Fluoromedroxyprogesterone acetate (FMPA) is a synthetic analog of medroxyprogesterone acetate (MPA). FMPA exhibited more potent anti-tumor and anti-angiogenic activities in some assay systems than the parent agent, MPA. Exudative age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV). Anecortave acetate, an angiostatic steroid, is clinically efficacious in patients with exudative AMD. Betamethasone is an anti-angiogenic steroid. Therefore, we examined the effects of FMPA, anecortave acetate and betamethasone on laser-induced CNV in rats. Anecortave acetate and betamethasone were included as positive controls. Crypton laser was applied to the fundus in Brown Norway rats. Laser photocoagulations were performed in each eye between the major retinal vessels of the superior retina. Subconjunctival injection of FMPA, anecortave acetate or betamethasone was performed once just after the photocoagulation (on day 0). The incidence of CNV formation was evaluated by fluorescein angiography (FAG) on day 14. On the next day, examination of the retinal function was performed by electro retinogram (ERG). Subconjunctival injection of FMPA at doses of 300, 1000 and 3000 μg/eye dose-dependently inhibited the incidence of CNV formation. Significant differences were observed at doses of 1000 and 3000 μg/eye of FMPA as compared with the control group. Anecortave acetate and betamethasone significantly inhibited the incidence of CNV formation. FMPA at the doses used in this study did not affect the retinal function in rats, as determined by ERG. FMPA appeared to be effective in a rat model of CNV, so it was demonstrated that FMPA might be useful in the treatment of AMD.
The vasorelaxant effect of cinnamaldehyde, one of the major oil components in Cinnamomi Cortex, was studied using isolated rat aorta. Cinnamaldehyde at final concentrations of 1 μM to 1 mM showed dose-dependent relaxation of the rat aorta contracted by treatment with prostaglandin F2α, norepinephrine or KCl. In addition, cinnamaldehyde relaxed prostaglandin F2α-precontracted aortic rings with endothelium and without endothelium, with the latter being significantly less sensitive than the former. Relaxation induced by cinnamaldehyde with endothelium was significantly inhibited by NG-nitro-L-arginine methyl ester (L-NAME), while nonselective cyclooxygenase inhibitor (indomethacin), β-adrenergic receptor blocker (propranolol), an inhibitor of phosphodiesterase (theophylline), a delayed rectifier K+ channel blocker (tetraethyl ammonium chloride), or an ATP-sensitive K+ channel blocker (glibenclamide) did not reduce the relaxation induced by cinnamaldehyde with endothelium treated by L-NAME. Conversely, aorta pretreatment with L-NAME and theophylline increased the relaxation by cinnamaldehyde significantly compared to aorta pretreatment with only L-NAME. Furthermore, cinnamaldehyde significantly inhibited Ca2+-induced contraction. These results suggested that the vasorelaxant effects of cinnamaldehyde were derived from both endothelium-dependent and -independent effects. Endothelium-dependent relaxation is affected by nitric oxide, and one of the mechanisms of endothelium-independent relaxation is thought to be influenced by the blocking of Ca2+ channels.
We previously reported that Smilacis chinae rhizome inhibits amyloid β protein (25—35) (Aβ (25—35))-induced neurotoxicity in cultured rat cortical neurons. The present study evaluated the neuroprotective effect of oxyresveratrol isolated from Smilacis chinae rhizome against Aβ (25—35)-induced neurotoxicity on cultured rat cortical neurons. Oxyresveratrol over the concentration range of 1—10 μM significantly inhibited 10 μM Aβ (25—35)-induced neuronal cell death, which was measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Oxyresveratrol (10 μM) inhibited 10 μM Aβ (25—35)-induced elevation of cytosolic calcium concentration ([Ca2+]c), which was measured by a fluorescent dye, Fluo-4 AM. Oxyresveratrol (1, 10 μM) also inhibited glutamate release into medium and reactive oxygen species (ROS) generation induced by 10 μM Aβ (25—35). These results suggest that oxyresveratrol prevents Aβ (25—35)-induced neuronal cell damage by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release and ROS generation. Furthermore, these effects of oxyresveratrol may be associated with the neuroprotective effect of Smilacis chinae rhizome.
To investigate the protective effect of ginsenoside Re (Re) against cerebral ischemia–reperfusion injury, adult male Wistar rats weighing 250—300 g were subjected to either sham surgery or middle cerebral artery occlusion (MCAO) for 2 h of brain ischemia and 2 h reperfusion. A fluorescence polarization assay was carried out for membrane fluidity of brain mitochondria. Lipid peroxidation [malondiadehyde (MDA) formation], superoxide dismutase (SOD) and glutathion peroxidase (GSH-Px) of rat brain were estimated by fluorometric methods. It was observed that Re (5, 10, 20 mg kg−1p.o. pretreatment for 7 d, once a day) significantly improved the fluidity of mitochondrial membranes as demonstrated by a reduction of average microviscosity, ameliorated lipid peroxidation by raising the activities of SOD and GSH-Px, and reduced the content of MDA in rat brain. This study demonstrated a direct protective effect of Re against cerebral ischemia–reperfusion injury.
Cry-consensus peptide (CCP) is a newly designed peptide for peptide-based immunotherapy of Japanese cedar pollinosis but its mechanism of efficacy is unknown. We investigated the effect of CCP on Cry j 1-specific Th1/Th2 response in a mice model. Subcutaneous injection of CCP decreased Cry j 1-specific IgE and IgG1 in blood slightly, but the IgG2a level was increased significantly in a dose dependent manner. Splenocytes from these mice were stimulated with Cry j 1 in vitro. This inhibited IL-4, IL-5 and IL-10 secretion significantly, but IFN-γ secretion was increased. In vitro CCP stimulation of splenocytes from Cry j 1-sensitized mice induced more marked Th1-predominancy of cytokine production than native allergen stimulation. Taken together, these data suggest that one of the mechanisms of CCP is dependent on the modulation of the antigen-specific Th1/Th2 response.
Recent studies have shown that Panax ginseng has a variety of beneficial effects on the cardiovascular systems. Homocysteine (Hcy), which is derived from L-methionine (Met), has been closely associated with the increased risk of cardiovascular diseases. In the present study, we examined whether in vivo long-term administration of ginseng saponins (GS), active ingredients of Panax ginseng, attenuate adverse vascular effects associated with chronic Met-induced hyperhomocysteinemia (H-Hcy). We found that plasma Hcy level, which was measured after 30 and 60 d, in GS (100 mg/kg)+Met co-administration group was significantly reduced when it was compared with Met alone treatment group. We could also observe the alleviation of endothelial damages of aortic artery vessels in GS (100 mg/kg)+Met co-administration group compared with Met alone treatment group. We compared aortic vasocontractile and vasodilatory responses between Met alone and GS (100 mg/kg)+Met co-treatment groups. We found that norepinephrine-induced vasocontractile responses were greatly decreased in GS (100 mg/kg)+Met co-treatment group and that carbachol-induced dilatory responses were greatly enhanced in GS (100 mg/kg)+Met co-administration groups as compared with Met alone treatment group. The present results indicate that in vivo long-term administration of GS attenuates adverse vascular effects associated with chronic Met-induced H-Hcy in rats.
To understand the relationship between the metabolites and estrogenic activity of the main isoflavones puerarin and daidzin of the rhizome of Pueraria thunbergiana (PT, family Leguminosae), PT and its isoflavones were transformed by human intestinal bacteria and their estrogenic effects were investigated. All human fecal specimens hydrolyzed puerarin and daidzin to daidzein, but their hydrolyzing activities varied dependenting on the individuals. All intestinal bacteria isolated from human also hydrolyzed daidzin to daidzein, but a few bacteria transformed puerarin to daidzein. When the estrogenic effect of PT, puerarin and daidzin was compared with those of their metabolites, the metabolites more potently increased proliferation of MCF-7 cells than PT, puerarin and daidzin. The metabolite daidzein also potently increased estrogen-response c-fos mRNA and PR protein expressions. These findings suggest that intestinal bacteria, which can hydrolyze puerarin and/or daidzin, may activate a potent estrogenic activity of PT.
A study was conducted to determine whether the ethanol extract of the roots of Brassica rapa (EBR) ameliorates cisplatin-induced nephrotoxicity in terms of oxidative stress, as characterized by lipid peroxidation, reactive oxygen species (ROS) production, and glutathione (GSH) depletion in LLC-PK1 cells. Pretreatment of cells with EBR prevented cisplatin-induced decreases in cell viability and cellular GSH content. The effect of EBR was then investigated in rats given EBR for 14 d before cisplatin administration. A single dose of cisplatin (7 mg/kg, i.p.) caused kidney damage manifested by an elevation in blood urea nitrogen (BUN), serum creatinine, and urine lactate dehydrogenase (LDH) levels. Also, renal tissue from cisplatin-treated rats showed a significant increase in malondialdehyde (MDA) production, and in the activities of aldehyde oxidase (AO) and xanthine oxidase (XO). Moreover, a significant decrease in the activities of antioxidant enzymes, such as, glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) was observed in cisplatin-treated rats versus saline-treated normal group. In contrast, rats given EBR showed lower blood levels of BUN and creatinine, and of urinary LDH. Moreover, EBR prevented the rise of MDA production and the induction of AO and XO activities. This extract also recovered the reduced activities of GPx, SOD and CAT. Taken together, our data indicate that the ethanol extract of the roots of Brassica rapa (EBR) has a protective effect against cisplatin-induced nephrotoxicity because it attenuates oxidative stress.
In China, the collection of wild Glycyrrhiza uralensis, one of the raw materials of Chinese licorice, has been restricted to prevent desertification. To compensate for the reduced supply of wild Glycyrrhiza plants, cultivation programs of G. uralensis have been initiated in eastern Inner Mongolia. The goal of the present study was to compare the chemical and pharmacological properties of cultivated G. uralensis roots to those of licorice prepared from wild Glycyrrhiza plants. The antispasmodic effect of boiled water extract of 4-year-old cultivated G. uralensis roots and licorice on carbachol-induced contraction in mice jejunum was similar (ED50: 134±21 μg/ml vs. 134±16 μg/ml). In addition, glycycoumarin content, which is an antispasmodic and species-specific ingredient of G. uralensis, was similar when comparing the boiled water extracts of 4-year-old cultivated roots and licorice (0.10±0.02% vs. 0.10±0.06%). These data suggest that cultivated G. uralensis roots may be an adequate replacement for the generation of licorice in the context of the restriction of wild Glycyrrhiza plant collection.
The young sticks and leaves of Sauropus androgynus (SA) that had been used as a health food for body weight reduction, led to an outbreak of obliterative bronchiolitis in Taiwan. This study tested the toxicity and anti-obesity features of the SA-isolated compound, 3-O-β-D-glucosyl-(1→6)-β-D-glucosyl-kaempferol (GGK), on male Wistar rats receiving 6 or 60 mg/kg of GGK orally as well as partial purified EtOAc and n-BuOH fractions of SA extract daily for 28 d. Sixty milligrams per kilogram GGK treatment significantly reduced food intake in rats by 15% (p<0.05). The reduced food intake corresponded to decreases in body weight in the high or low dose GGK groups, as compared to the control groups. The serum levels of free triglyceride significantly decreased in GGK-treated rats. GGK treatment led to succesive reductions in daily food intake and body weight without obvious histopathological changes in Wistar rats. Thus, GGK may be potentially to be developed as a safe and novel compound for anti-obesity treatment.
In autumn 2004, many Japanese patients with renal failure developed cryptogenic encephalopathy by consuming sugihiratake mushroom, a Japanese delicacy. To elucidate the relationship between the cryptogenic cases and this mushroom, we conducted a multivariate analysis of metabolites in ‘Probably Toxic’ sugihiratake collected from the area of encephalopathy outbreaks, and ‘Probably Safe’ sugihiratake collected from unaffected areas using UPLC/ToF MS. The results indicate that the presence of milligram quantities of vitamin D-like compounds per 10 g of dried sugihiratake from the areas of encephalopathy outbreaks. Two hypotheses to induce the encephalopathy are proposed: the found metabolites are (1) vitamin D agonists, which induce acute and severe hypercalcemia and/or hyperammonemia and/or vitamin D toxicity, or (2) vitamin D antagonists, which induce acute and severe hypocalcemia.
P-Glycoprotein (ABCB1-type P-gp), a membrane protein encoded by the multi drug resistant gene (MDR1), expressing on the blood brain barrier protects the brain from many drugs including dexamethasone. Psychiatric disorders, schizophrenia and depression, have known to have abnormal hypothalamus-pituitary-adrenal (HPA) activity, which is assessed by non-suppression of cortisol in dexamethasone suppression test. The poor response to dexamethasone in these patients' population suggested the impaired activity on dexamethasone penetration into the brain via P-gp, which was associated with MDR1 polymorphisms. We, therefore, examined five SNPs of the MDR1 gene, −1517 T>C (promoter), −41 A>G (intron −1), −129 T>C (exon 1b), 2677 G>A,T (exon 21) and 3435 C>T (exon 26), in Japanese patients with schizophrenia (n=121) and mood disorders (n=62), and compared with the control subjects (n=160). The frequency of MDR1 mutant alleles at −1517, −41 and −129 in patients with mood disorders was significantly lower (2.4, 5.6, 2.4%, respectively) than those of controls (7.8, 13.7, 7.8%, respectively) (p<0.05). The frequencies of MDR1 2677 G/A and A/A genotype in mood disorders was significantly higher (17.7, 6.5%, respectively) than controls (11.2, 0%, respectively) (p<0.05). The 2677A allele frequency in mood disorders (20.2%) was significant higher than controls (10.9%) (p<0.05). Haplotype of 129-2677-3435 (T-A-C) in mood disorders was significantly higher (14.4%) than controls (8.0%) (p<0.05). There was no significant difference in allele and genotype frequencies between the patients with schizophrenia and controls. These findings suggested that predispose to mood disorders, not schizophrenia, was associated with possible alteration of P-gp activities corresponding MDR1 polymorphism at least partly.
In a prior study, we reported on a significant decrease in calpain10 gene expression in white blood cells (WBC) as well as the major insulin-target tissues including liver and adipose tissue, before the onset of diabetes in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. In this study, we extended our hypothesis that some type 2 diabetes mellitus (NIDDM) susceptible genes are up/down-regulated before the onset in WBC of OLETF rats, reflecting their up/down-regulation in major insulin-target tissues, such as the liver. We tested this hypothesis using rat cDNA microarrays. The findings show that 1080 genes are up/down-regulated by more than 2-fold compared to the controls, Long-Evans Tokushima Otsuka rats, before the onset in WBC and liver under fasted or insulin administered condition. Fifty-seven of the 1080 genes were up/down-regulated in both WBC and the liver. More than half have been reported to NIDDM susceptible genes and the remainder have not been reported to be related to NIDDM. These results indicate that there some NIDDM related genes are up/down-regulated in WBC before the onset of diabetes.
The population pharmacokinetic parameters of mizoribine in healthy subjects were estimated using a nonlinear mixed effects model (NONMEM) program. Pharmacokinetic data for population analysis were obtained in the previous study, in which 24 healthy Caucasian male subjects participated in a single-dose (3, 6, 9, 12 mg/kg) study, and 12 subjects participated in a multiple-dose (6, 12 mg/kg/d) study. The mean value of the absorption lag time, absorption rate constant (KA), and apparent distribution volume (V/F) was estimated to be 0.349 h, 0.869 h−1, and 0.834 l/kg, respectively. Oral clearance (CL/F) was modeled with creatinine clearance (CLcr), and the mean value was estimated to be 1.93·CLcr l/h. In addition, pharmacokinetic parameters in individual 36 subjects were obtained from population estimates according to Bayes' theorem. Pharmacokinetic parameters (KA, V/F, and CL/F) in the single-dose study were almost constant at a dose range of 3—12 mg/kg, and were similar to those in the multiple-dose study. These findings indicated that the pharmacokinetics of mizoribine is well described by a simple one-compartment model with first-order absorption.
Human ABC transporter P-glycoprotein (P-gp/ABCB1) encoded by the multidrug resistance (MDR1) gene is recognized as one of the most important factors regulating pharmacokinetics of a number of clinically important drugs because of its function of extruding a wide range of structurally unrelated amphiphilic and hydrophobic drugs from the inside to the outside of cells in an ATP-driven mechanism. In the present study, we have evaluated the high-speed ATPase activity assay method by comparing with in vitro transport assay systems using MDR1-transfected MDR1-MDCK cells. Since substrate drugs were found to interfere with the photometric detection of inorganic phosphate (Pi) that was liberated according to the hydrolysis of ATP to ADP in ATPase activity assay, at first, a method in which the amount of Pi can be calculated correctly. Results demonstrate that the kinetic parameters obtained in ATPase activity assay are not necessarily correspond with those in in vitro transport assay, suggesting that these methods might detect the different processes of drug-P-gp interaction. The combining of the ATPase activity assay and in vitro transport technologies provides us the insight into mechanisms of the membrane transport of drugs by P-gp.
In order to assess the beneficial mechanism of the concomitant use of imipenem (IPM) with piperacillin (PIPC) for the treatment of serious infectious diseases such as sepsis, the effects of PIPC on the uptake of IPM by rat renal cortical slices and on the plasma concentrations of IPM after intravenous infusion to rabbits were studied. The uptake of IPM by the rat renal cortical slices was significantly inhibited by p-aminohippurate, probenecid and PIPC whereas the uptake of PIPC by the slices was slightly decreased in the presence of IPM. When IPM was administered together with PIPC by 1-h infusion, the plasma concentrations of IPM were significantly increased during the infusion. These results imply that PIPC possibly interferes with the renal transport of IPM mediated by an organic anion transporter across the renal basolateral membranes, which leads to a longer period above the minimum inhibitory concentrations of IPM.
Lu xian cao (Hebra pyrolae) is a traditional Chinese medicine used for the treatment of several major diseases. Chimaphilin has been demonstrated one of the major active components in Lu xian cao by modern pharmacological studies. In this study, a liquid chromatography-mass spectrometry method for the determination of chimaphilin in rat plasma was developed and validated. The separation was carried out on a C18 column using methanol and water as mobile phase after the plasma sample was extracted with diethyl ether. Atmospheric pressure chemical ionization in negative ion mode and selected ion monitoring method were developed to determine [M]− at 186 and 210 for chimaphilin and benzil (internal standard), respectively. The lower limit of quantitation was 10 ng/ml and the calibration curve was linear (r>0.9964) over the concentration range 10—1000 ng/ml. The method was demonstrated reproducible and reliable with intra-day precision <11.5%, inter-day precision <7.6%, accuracy in the range of 88.4—113.0%, and mean extraction recovery excess of 83.0%, which were all calculated from the quality control samples at concentrations of 20, 100, and 500 ng/ml. The method was successfully applied to pharmacokinetic study of chimaphilin in rat plasma following oral administration of a 30-mg/kg dose of chimaphilin in Lu xian cao decoction to male Wistar rats.
The purpose of this study was to assess the impact by switching co-administered triazole antifungal agent from fluconazole (FCZ) to voriconazole (VCZ) on the blood concentration of tacrolimus (FK506) in patients receiving allogeneic hematopoietic stem cell transplantation. We performed a retrospective study presented as case reports. The blood concentration of FK506 was increased after the switch from FCZ to VCZ, resulting in increase of the concentration/dose (C/D) ratio of FK506. Thus, the mean C/D ratios of FK506 with oral administration was surprisingly increased over 4.5-fold after the switch. Therefore, it was necessary to reduce the FK506 dose when co-administered FCZ is switched to VCZ. We should be careful when interpreting the results of these case reports; however, in some patients, it is recommended that the dose of FK506 be reduced to one-fifth after the switch.
The effect of young and mature persimmon fruits on lipid metabolism was investigated in a diet-induced murine obesity model. A commercially purchased high fat diet (Quick Fat, CLEA Japan) was used as the basal diet. Dried and powdered young and mature fruits of two breeds of persimmon, Fuyu-kaki and Hachiya-kaki, were added to the basal diet at a concentration of 10%, respectively. Male C57BL/6 mice (n=4) were divided into five groups and fed the basal diet or one of the persimmon-supplemented basal diets ad libitum for 14 weeks. Diets supplemented with both types of young fruit significantly reduced the rise in plasma lipids, including total cholesterol (p<0.005), triglyceride (p<0.05), and LDL cholesterol (p<0.05), and the effect was almost equal between the two breeds. Real-time RT-PCR revealed that both of these young fruit-supplemented diets equally up-regulated expression of the cholesterol 7 alpha-hydroxylase (CYP7A1) gene in the liver by about three-fold (p<0.05). CYP7A1 plays an important role in maintaining cholesterol homeostasis by regulating bile acid synthesis, suggesting that increased conversion of cholesterol to bile acids may have caused the cholesterol-lowering effect of the young fruits. The results indicate that young persimmon fruits are beneficial in the development of preventive and therapeutic agents against dyslipidemia.
Ginsenosides comprise the major component of ginseng exhibit various types of biological activity, including antiinflammatory and antitumor effects. In these pharmacological actions, it is thought that these activities are carried out by the metabolites of ginsenosides metabolized by human intestinal microflora. It has also been reported that their clinical efficacy varies with the hydrolyzing potential of the components of the intestinal microflora. We tried to develop a process for metabolizing ginsenosides to compound K using food-grade enzymes, which can be used commercially. Among these, Pectinex proved to be the most effective mediator of the catabolism of ginsenosides to compound K. The optimal conditions for this biotransformation were determined to be as follows: 10 to 15% rootlet ginseng, pH 5, 50 °C, and 2 to 3 d of incubation, to yield 20.0 mg of compound K/g of rootlet ginseng. We suggest that the metabolism of ginseng to compound K in the presence of Pectinex has many advantages over previous methods, in respects of use of raw, non-extracted rootlet ginseng, which do not require more organic solvents and evaporation apparatus. Potential metabolites PG1, PG2, PG3, and PG4 were detected in Pectinex-treated rootlet ginseng using by TLC and HPLC and, among them, PG4 was identified as compound K by TLC, HPLC, and MS. Additional studies will be carried out to determine the structure of these metabolites of ginseng and to understand the relationship between their structures and activities.