Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 41 , Issue 4
Showing 1-29 articles out of 29 articles from the selected issue
Regular Articles
  • Junko Kimura, Kosuke Shimizu, Koji Kajima, Akihito Yokosuka, Yoshihiro ...
    2018 Volume 41 Issue 4 Pages 451-457
    Published: April 01, 2018
    Released: April 01, 2018
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    Alzheimer’s disease (AD) is the most common cause of dementia, with progressive memory impairment. Recently, neprilysin, a β-amyloid (Aβ)-degrading enzyme has become featured as a drug target for AD. Previously, we identified nobiletin from citrus peels as a natural compound possessing anti-dementia activity. In addition, we demonstrated that nobiletin improved memory in memory-impaired animals and, further, that Aβ levels were markedly decreased in the brains of these animals. We demonstrated in vitro that nobiletin up-regulates neprilysin expression and activity in human neuroblastoma cells. However, the action of nobiletin with regard to Aβ degradation under in vitro AD pathological conditions remains unclear. In this study, we examined whether nobiletin could enhance the degradation of intra- and extracellular Aβ using human induced pluripotent stem cell-derived AD model neurons, which generate an excess of Aβ1–42 due to the familial AD presenilin-1 mutation. The neurons were treated in the presence or absence of nobiletin. The results of real-time quantitative RT-PCR indicated that neprilysin mRNA levels were significantly up-regulated by nobiletin. Furthermore, immunostaining with an anti-Aβ antibody revealed that nobiletin substantially reduced the intraneuronal content of Aβ. Interestingly, the results of Aβ1–42 immunoassays confirmed that nobiletin also significantly decreased the levels of Aβ1–42 released into the cellular medium. These results suggest that nobiletin enhanced the reduction of intra- and that extracellular Aβ levels under AD pathologic conditions, and this is associated with the up-regulation of neprilysin expression. Collectively, nobiletin appears to be a promising novel prophylactic seed drug or functional food for AD.

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  • Ren Wu, Ding Li, Qi Tang, Wanchun Wang, Guangrong Xie, Pengcheng Dou
    2018 Volume 41 Issue 4 Pages 458-464
    Published: April 01, 2018
    Released: April 01, 2018
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    Osteosarcoma (OS) is a typical bone cancer, and most frequently used cancer treatments for OS are limited due to severe drug-related toxicities. Wasp venoms contain functional components that may offer pharmaceutical components for the treatment of cancers. This study aimed to isolate and characterize a novel peptide (venom anti-cancer peptide 1, VACP1) derived from the wasp venom of Vespa ducalis SMITH. Toxins from Vespa ducalis crude venom were separated by gel filtration and purified by C18 reverse-phase HPLC. As examined by Edman degradation, the amino acid sequence of VACP1 is AQKWLKYWKADKVKGFGRKIKKIWFG. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed that VACP1 inhibited the cell proliferation of MG-63, U-2 OS and Saos-2 cells. Furthermore, annexin V and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining revealed that VACP1 could induce the apoptosis of OS cell lines. In addition, VACP1 increased the protein levels of cleaved poly ADP-ribose polymerase (PARP), caspase 3, but decreased B-cell lymphoma 2 (Bcl-2). Apoptotic signaling pathway screening in MG-63 cells via an antibody array revealed that VACP1 activated the p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways. The present study demonstrates that VACP1 potently suppressed cell proliferation and induced the cell apoptosis of OS cells by inducing the activation of the p38 MAPK and JNK signaling pathways, suggesting that VACP1 is a promising agent for OS therapy.

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  • Hiroshi Shimamura, Hiroko Yamada, Natsumi Okada, Takeshi Uchikura, Kat ...
    2018 Volume 41 Issue 4 Pages 465-469
    Published: April 01, 2018
    Released: April 01, 2018
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    The dosage and frequency of lamotrigine administration for each patient must be prescribed carefully according to the disease, age, concomitant medications, and administration period. According to Pharmaceuticals and Medical Devices Agency (PMDA) reports, either the number of patients with adverse events caused by the inappropriate use of lamotrigine or the ratio of the patients who have benefited from the Relief System for Adverse Drug Reactions did not change even after the revision of the package insert in 2015. The procedure for auditing lamotrigine prescriptions was standardized to improve patient safety in our hospitals. The efficacy of standardization was evaluated by investigating the patients’ records and prescriptions. A total of 77 patients treated with lamotrigine were examined to evaluate the appropriateness of the dosage and frequency of administration as well as the number of prescription enquiries made by pharmacists retrospectively. In addition, the presence of adverse events such as skin rash was examined using medical records. The number of inappropriate cases found and the prescription questions asked by pharmacists during the 24-month period before and after standardization were compared. The rate of inappropriate prescriptions after standardization was significantly lower than that before (12.1 vs. 29%; p<0.05). The rate of prescription questions raised after standardization was significantly higher than that before (37.0 vs. 1.7%; p<0.05). Moreover, there was no adverse event after standardization. Our standardization practice was effective to easily identify inappropriate prescriptions and provide physicians with the appropriate dosage or frequency of administration for the patients.

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  • Quan Feng Liu, Youngjae Jeon, Yung-wei Sung, Jang Ho Lee, Haemin Jeong ...
    2018 Volume 41 Issue 4 Pages 470-477
    Published: April 01, 2018
    Released: April 01, 2018
    [Advance publication] Released: February 03, 2018
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    The Nardostachys jatamansi DC (NJ) root has been used as a sedative or analgesic to treat neurological symptoms and pain in traditional Korean medicine. Here, we investigate the potential effects of NJ on Alzheimer’s disease (AD) and reveal the molecular mechanism through which NJ exerts its effects. The neuroprotective effect of the NJ root ethanol extract against β amyloid (Aβ) toxicity was examined in vitro using a cell culture system and in vivo using a Drosophila AD model. The NJ extract and chlorogenic acid, a major component of NJ, inhibited Aβ-induced cell death in SH-SY5Y cells. Moreover, the NJ extract rescued the neurological phenotypes of the Aβ42-expressing flies (decreased survival and pupariation rate and a locomotor defect) and suppressed Aβ42-induced cell death in the brain. We also found that NJ extract intake reduced glial cell number, reactive oxygen species level, extracellular-signal-regulated kinase (ERK) phosphorylation, and nitric oxide level in Aβ42-expressing flies, without affecting Aβ accumulation. These data suggest that the neuroprotective activity of NJ might be associated with its antioxidant and anti-inflammatory properties, as well as its inhibitory action against ERK signaling; thus, NJ is a promising medicinal plant for the development of AD treatment.

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  • Yuanlong Li, Sen Lin, Chang Xu, Peng Zhang, Xifan Mei
    2018 Volume 41 Issue 4 Pages 478-486
    Published: April 01, 2018
    Released: April 01, 2018
    [Advance publication] Released: January 23, 2018
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    High level apoptosis induced by spinal cord injury (SCI) evokes serious damage because of the loss and dysfunction of motor neurons. Our previous studies showed that inhibition of autophagy evokes the activation of apoptosis. Interestingly, Baicalein, a medicine with anti-apoptosis activity that is derived from the roots of herb Scutellaria baicalensis, largely induces autophagy by activating phosphatidylinositol 3-kinase (PI3K). In this study, we investigated the effects of intraperitoneal injection of Baicalein on autophagy and apoptosis in SCI mice and evaluated the relationship between autophagy and apoptosis. We demonstrated that Baicalein promoted the functional recovery of motor neurons at 7 d after SCI. In addition, Baicalein enhanced neuronal autophagy and the autophagy-related factor PI3K, while inhibiting the p62 protein. Baicalein treatment decreased neuronal apoptosis at 7 d after SCI. Moreover, when inhibiting autophagy, apoptosis was upgraded by Baicalein treatment after injury. Thus, Baicalein attenuated SCI by inducing autophagy to reduce apoptosis in neurons potentially via activating PI3K.

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  • Babita Shashni, Shinya Ariyasu, Reisa Takeda, Toshihiro Suzuki, Shota ...
    2018 Volume 41 Issue 4 Pages 487-503
    Published: April 01, 2018
    Released: April 01, 2018
    [Advance publication] Released: January 13, 2018
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    Detection of anomalous cells such as cancer cells from normal blood cells has the potential to contribute greatly to cancer diagnosis and therapy. Conventional methods for the detection of cancer cells are usually tedious and cumbersome. Herein, we report on the use of a particle size analyzer for the convenient size-based differentiation of cancer cells from normal cells. Measurements made using a particle size analyzer revealed that size parameters for cancer cells are significantly greater (e.g., inner diameter and width) than the corresponding values for normal cells (white blood cells (WBC), lymphocytes and splenocytes), with no significant difference in shape parameters (e.g., circularity and convexity). The inner diameter of many cancer cell lines is greater than 10 µm, in contrast to normal cells. For the detection of WBC having similar size to that of cancer cells, we developed a PC software “Cancer Cell Finder” that differentiates them from cancer cells based on brightness stationary points on a cell surface. Furthermore, the aforementioned method was validated for cancer cell/clusters detection in spiked mouse blood samples (a B16 melanoma mouse xenograft model) and circulating tumor cell cluster-like particles in the cat and dog (diagnosed with cancer) blood samples. These results provide insights into the possible applicability of the use of a particle size analyzer in conjunction with PC software for the convenient detection of cancer cells in experimental and clinical samples for theranostics.

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  • Yoichi Sunagawa, Nobuko Okamura, Yusuke Miyazaki, Kana Shimizu, Mai Ge ...
    2018 Volume 41 Issue 4 Pages 504-509
    Published: April 01, 2018
    Released: April 01, 2018
    [Advance publication] Released: February 06, 2018
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    Neck and shoulder stiffness is a typical subjective symptom in developed countries. This stiffness is caused by factors such as muscle tension and poor blood flow, leading to reduce work efficiency and diminish QOL. NKCP®, a natto-derived dietary food supplement whose main component is bacillopeptidase F, has antithrombotic, fibrinolytic, and blood viscosity-lowering effects. Here, we investigated the effect of NKCP® on neck and shoulder stiffness in a double-blind placebo-controlled randomized crossover study. Thirty subjects with neck and shoulder stiffness were randomly divided into 2 groups and ingested 250 mg of NKCP® or placebo daily for 4 weeks. Headache score significantly improved in the NKCP® group compared to the placebo group. Moreover, NKCP® significantly improved the score of visual analogue scale for neck and shoulder stiffness and pain, reduced muscle stiffness of the neck, and increased the skin surface temperature of neck and shoulders, compared to before ingestion. No adverse effects were observed during this study. These results suggest that NKCP® may alleviate headaches and chronic neck and shoulder stiffness and pain.

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  • Hiroko Tokumoto, Hiroko Shimomura, Takashi Hakamatsuka, Yoshihiro Ozek ...
    2018 Volume 41 Issue 4 Pages 510-523
    Published: April 01, 2018
    Released: April 01, 2018
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    Microscopic examination of crude drug components has been the traditional method to identify the origin of biological materials. For the identification of components in a given mixture via microscopy, standard reference photographs of fragments derived from different organs and tissues of individual species are required. In addition to these reference photographs, a highly observant eye is needed to compare the morphological characteristics observed under the microscope with those of the references and to then identify the origin of the materials. Therefore, if other indexes are available to be coupled with microscope examination, the accuracy of identification would be significantly improved. Here, we prepared standard reference photographs for microscopic examination to identify powdered and fragmented materials in the crude drug “Quanxie” derived from individual organs of dried scorpion (Buthus martensii KARSCH). Since a remarkable characteristic of scorpion bodies is that they fluoresce under UV light, two methods to identify “Quanxie” were established, including fluorescence fingerprint analysis and microscopic fluorescent luminance imaging analysis. In the former, at least 0.1 g of powered materials was used, which could be recovered after the measurement, and in the latter, only small amounts of powders were used for microscopic examinations. Both methods could distinguish powders of “Quanxie” from those of other micro-morphologically similar crude drugs, namely, “Chantui,” “Sangpiaoxiao,” and “Jiangcan.” The combination of these methods should improve the swiftness and accuracy of “Quanxie” identification.

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  • Kensuke Nakajima, Shigeru Oiso, Hiroko Kariyazono
    2018 Volume 41 Issue 4 Pages 524-529
    Published: April 01, 2018
    Released: April 01, 2018
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    Ghrelin is an orexigenic peptide hormone produced in the stomach. The major active form is octanoylated ghrelin, which is modified with an n-octanoic acid at the serine-3 residue. Inhibition of octanoylated ghrelin production is useful for the prevention and improvement of obesity. We previously developed a cell-based assay system employing a ghrelin-expressing cell line, AGS-GHRL8, and found various compounds that decreased octanoylated ghrelin levels using this system. (−)-Epigallocatechin-3-O-gallate (EGCG) is a bioactive catechin in green tea and reportedly has an anti-obesity effect; however, it remains unclear whether EGCG inhibits octanoylated ghrelin production. Therefore, in this study, we investigated the effect of EGCG on octanoylated ghrelin levels in AGS-GHRL8 cells and C57BL/6J mice. EGCG significantly reduced the octanoylated ghrelin level in AGS-GHRL8 cells. In mice, three days of treatment with TEAVIGO®, which contains 97.69% EGCG, lowered the plasma octanoylated ghrelin level by 40% from that in control mice. In addition, TEAVIGO® reduced the mRNA expression of ghrelin and prohormone convertase 1/3, an enzyme responsible for the processing of proghrelin to mature ghrelin, in the mouse stomach, suggesting that the reduced expression of these genes may contribute to the inhibition of octanoylated ghrelin production. These results suggest a decrease in the octanoylated ghrelin level to be involved in the anti-obesity effect of EGCG, which thus has potential for the development of anti-obesity agents with ghrelin-lowering effect.

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  • Chen Zhong, Liyong Pu, Mingming Fang, Jianhua Rao, Xuehao Wang
    2018 Volume 41 Issue 4 Pages 530-535
    Published: April 01, 2018
    Released: April 01, 2018
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    All-trans retinoic acid (ATRA) has been proved to protect liver from ischemia/reperfusion (IR) injury, however, its mechanism is still unclear. This study is to investigate the mechanism of effect of ATRA on innate immunity in mice liver IR injury. Before operation, mice were gavaged by ATRA at 15 mg/kg/d for two weeks, and then the liver was underwent 70% ischemia (90 min) and reperfusion (6 h). Liver function was assessed by serum alanine aminotransferase (sALT), serum aspartate aminotransferase (sAST). Real-time PCR and Western blot were to detect the level of mRNA and protein. In vitro, RAW264.7 macrophages were treatment with ATRA (1 µM) or LE540 (5 µM, a retinoic acid receptor α (RARα) receptor antagonist) before lipopolysaccharide (100 ng/mL) stimulation. In vivo, ATRA protected the liver from IR injury by improving hepatocellular function (sALT and sAST), decreasing cell apoptosis and inhibiting inflammatory response (i.e., the level of toll-like receptor 4, transcription factor nuclear factor-κBp65, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α). When RARα was blocked by LE540 in RAW264.7 macrophages, the inflammatory cytokines were enhancing, along with a decline of Akt phosphorylation but Forkhead box o (Foxo) 1, compared with the ATRA group. In summary, ATRA regulates in part the innate immunity to protect liver from IR injury by RARα/Akt/Foxo1 pathway.

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  • Masafumi Noda, Masaya Shiraga, Takanori Kumagai, Narandalai Danshiitso ...
    2018 Volume 41 Issue 4 Pages 536-545
    Published: April 01, 2018
    Released: April 01, 2018
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    Lactobacillus plantarum SN35N, which has been previously isolated from pear, secretes exopolysaccharide (EPS). The aim of the present study is to characterize the EPS chemically and to find the EPS-biosynthesizing gene cluster. The present study demonstrates that the strain produces an acidic EPS carrying phosphate residue, which is composed of glucose, galactose, and mannose at a molecular ratio of 15.0 : 5.7 : 1.0. We also show that acidic EPS strongly inhibits the catalytic activity of hyaluronidase (EC 3.2.1.35), promoting an inflammatory reaction. In the present study, we also determined the complete genome sequence of the SN35N strain, demonstrating that the genome is a circular DNA with 3267626 bp, and the number of predicted coding genes is 3146, with a GC content of 44.51%. In addition, the strain harbors four plasmids, designated pSN35N-1, -2, -3, and -4. Although four EPS-biosynthesizing genes, designated lpe1, lpe2, lpe3, and lpe4, are present in the SN35N chromosomal DNA, another EPS gene cluster, lpe5, is located in the pSN35N-3 plasmid, composed of 35425 bp. EPS low-producing mutants, which were obtained by treating SN35N cells with novobiocin, lost the lpe5 gene cluster in the plasmid-curing experiment, suggesting that the gene cluster for the biosynthesis of acidic EPS is present in the plasmid. The present study shows the chemical characterization of the acidic EPS and its inhibitory effect to the hyaluronidase.

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  • Noriyasu Kamei, Ai Yamaoka, Yukiko Fukuyama, Rei Itokazu, Mariko Taked ...
    2018 Volume 41 Issue 4 Pages 546-554
    Published: April 01, 2018
    Released: April 01, 2018
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    To overcome the difficulty in delivery of biopharmaceuticals such as peptides and proteins to the brain, several approaches combining the ligands and antibodies targeting the blood–brain barrier (BBB) have been tried. However, these are inefficient in terms of their permeability through the BBB and structural modification of bioactive drugs. In the present study, we therefore examined the usefulness of a noncovalent method using the cell-penetrating peptides (CPPs) such as octaarginine (R8) as a suitable brain delivery strategy for biopharmaceuticals. A safety examination using microvascular endothelial model bEnd.3 cells clarified that R8 was the safest among the CPPs tested in this study. The cellular uptake study demonstrated that coincubation with R8 enhanced the uptake of model peptide drug insulin by bEnd.3 cells in a concentration-dependent and a temperature-independent manner. Furthermore, an in vivo study with rats showed that the accumulation of insulin in the deeper region of the brain, i.e., hippocampus, significantly increased after the intravenous coadministration of insulin with D-R8 without altering the insulin disposition in plasma. Thus, the present study provided the first evidence suggesting that the noncovalent method with CPPs is one of the strategic options for brain delivery of biopharmaceuticals via intravenous injection.

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  • Katsunori Tsuda, Licht Miyamoto, Shuichi Hamano, Yuri Morimoto, Yumi K ...
    2018 Volume 41 Issue 4 Pages 555-563
    Published: April 01, 2018
    Released: April 01, 2018
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    Artemisinin was discovered in 1971 as a constituent of the wormwood genus plant (Artemisia annua). This plant has been used as an herbal medicine to treat malaria since ancient times. The compound artemisinin has a sesquiterpene lactone bearing a peroxide group that offers its biological activity. In addition to anti-malarial activity, artemisinin derivatives have been reported to exert antitumor activity in cancer cells, and have attracted attention as potential anti-cancer drugs. Mechanisms that might explain the antitumor activities of artemisinin derivatives reportedly induction of apoptosis, angiogenesis inhibitory effects, inhibition of hypoxia-inducible factor-1α (HIF-1α) activation, and direct DNA injury. Reactive oxygen species (ROS) generation is involved in many cases. However, little is known about the mechanism of ROS formation from artemisinin derivatives and what types of ROS are produced. Therefore, we investigated the iron-induced ROS formation mechanism by using artesunate, a water-soluble artemisinin derivative, which is thought to be the underlying mechanism involved in artesunate-mediated cell death. The ROS generated by the coexistence of iron(II), artesunate, and molecular oxygen was a hydroxyl radical or hydroxyl radical-like ROS. Artesunate can reduce iron(III) to iron(II), which enables generation of ROS irrespective of the iron valence. We found that reduction from iron(III) to iron(II) was activated in the acidic rather than the neutral region and was proportional to the hydrogen ion concentration.

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  • Yuki Araie, Yosuke Takahashi, Yuki Takahashi, Yoshinobu Takakura, Maki ...
    2018 Volume 41 Issue 4 Pages 564-569
    Published: April 01, 2018
    Released: April 01, 2018
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    A151 and other immunosuppressive oligodeoxynucleotides that act as Toll-like receptor (TLR) 9 antagonists are candidate agents for the treatment of autoimmune and inflammatory diseases in which TLR9 activation leads to harmful immune responses. Their efficient delivery to TLR9-positive target cells will increase their potency, but few attempts have been made to enhance their delivery. We previously reported that hexapod-like nanostructured DNA (hexapodna) enhanced the activity of immunostimulatory cytosine-phosphate-guanine (CpG) DNA by efficiently delivering it to immune cells. In this study, to enhance the immunosuppressive activity of A151, we designed a hexapodna containing six copies of the complementary sequence to A151. Structural analyses showed that A151-loaded hexapodna (supHexapodna) was obtained as designed. CpG 1668, which is a typical synthetic CpG DNA, induced tumour necrosis factor-α release from mouse macrophage-like RAW264.7 cells, and supHexapodna inhibited this more efficiently than A151. A flow cytometric analysis showed that the uptake of Alexa Fluor 488-labelled A151 by RAW264.7 cells significantly increased when it was incorporated into supHexapodna, whereas the uptake of Alexa Fluor 488-labelled CpG 1668 was hardly affected by A151 or supHexapodna. These results suggest that the hexapodna-mediated delivery of A151 can increase the potency of its TLR9-inhibitory activity towards immune cells.

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  • Halil Ibrahim Ciftci, Safiye Emirdag Ozturk, Taha F. S. Ali, Mohamed O ...
    2018 Volume 41 Issue 4 Pages 570-574
    Published: April 01, 2018
    Released: April 01, 2018
    [Advance publication] Released: January 30, 2018
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    The discovery of the chimeric tyrosine kinase breakpoint cluster region kinase-Abelson kinase (BCR-ABL)-targeted drug imatinib conceptually changed the treatment of chronic myelogenous leukemia (CML). However, some CML patients show drug resistance to imatinib. To address this issue, some artificial heterocyclic compounds have been identified as BCR-ABL inhibitors. Here we examined whether plant-derived pentacyclic triterpenoid gypsogenin and/or their derivatives show inhibitory activity against BCR-ABL. Among the three derivatives, benzyl 3-hydroxy-23-oxoolean-12-en-28-oate (1c) was found to be the most effective anticancer agent on the CML cell line K562, with an IC50 value of 9.3 µM. In contrast, the IC50 against normal peripheral blood mononuclear cells was 276.0 µM, showing better selectivity than imatinib. Compound 1c had in vitro inhibitory activity against Abelson kinase 1 (ABL1) (IC50=8.7 µM), the kinase component of BCR-ABL. In addition, compound 1c showed a different inhibitory profile against eight kinases compared with imatinib. The interaction between ATP binding site of ABL and 1c was examined by molecular docking study, and the binding mode was different from imatinib and newer generation inhibitors. Furthermore, 1c suppressed signaling downstream of BCR-ABL. This study suggests the possibility that plant extracts may be a source for CML treatment and offer a strategy to overcome drug resistance to known BCR-ABL inhibitors.

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  • Hao Zhang, Xiaoou Sun, Yanxiang Xie, Fang Tian, Hui Hu, Wen Tan
    2018 Volume 41 Issue 4 Pages 575-584
    Published: April 01, 2018
    Released: April 01, 2018
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    Previous reports have indicated that isosteviol sodium (STVNa) has neuroprotective effects against acute focal cerebral ischemia in rats; however, the exact underlying mechanisms and ideal treatment paradigm are not known. To find a reasonable method for STVNa administration and to determine its possible therapeutic mechanisms, we characterized the protective effects of single-dose and multiple-dose STVNa in cerebral ischemic/reperfusion (I/R) injury in rats. Single and multiple treatments with 10 mg/kg STVNa were administered intraperitoneally after injury to investigate its neuroprotective effects. Neurobehavioral deficits and infarct volume were assessed 7 d after ischemia. Both STVNa treatments reduced infarct volumes, improved neurological behaviors, preserved cellular morphology, enhanced neuronal survival, and suppressed cell apoptosis. Multiple treatments performed better than single treatment. Reactive astrogliosis was apparent at 7 d after injury and was significantly inhibited by multiple STVNa treatments but not single treatment. These results indicate that STVNa exerts neuroprotection by different mechanisms in the acute and delayed phases of I/R. Specifically, STVNa neuroprotection in the delayed phase of injury was found to be accompanied with the inhibition of astrogliosis.

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  • Sha-sha Li, Ao-lin He, Zhi-yong Deng, Qi-feng Liu
    2018 Volume 41 Issue 4 Pages 585-591
    Published: April 01, 2018
    Released: April 01, 2018
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    Ginsenoside-Rg1 (G-Rg1) is an agent isolated from Panax ginseng that exerts anti-fibrotic effects; however, the mechanism is still unclear. Herein, we investigated whether G-Rg1 administration can mitigate or reverse unilateral ureteral obstruction (UUO)-induced renal fibrosis by regulating the Klotho/transforming growth factor (TGF)-β1/Smad signaling pathway in rats. Sprague–Dawley male rats were subjected to UUO, and rats in the treatment group were administered G-Rg1 or G-Rg1 plus Klotho short hairpin RNA interference (shRNA), while rats in the control and model groups were administered vehicle for 14 d. Epithelial–mesenchymal transition (EMT) biomarkers and Klotho/TGF-β1 signaling molecules were examined by immunohistochemistry, quantitative real-time PCR and Western blotting. Immunohistochemistry showed that UUO induced increased pro-fibrotic TGF-β1 expression, overexpression of the mesenchymal marker, α-smooth muscle actin (α-SMA), and suppression of the epithelial marker, E-cadherin. Moreover, Western blotting analysis indicated that UUO promoted TGF-β1 and phosphorylated Smad3 (p-Smad3) expression (p<0.01), but blocked Klotho and Smad7 expression (p<0.01). After G-Rg1 administration, the UUO-induced TGF-β1 and p-Smad3 expression was suppressed (p<0.01), whereas the reduced Klotho and Smad7 expression was reversed (p<0.05), followed by amelioration of the EMT process. Intriguingly, the G-Rg1 effects were largely abrogated by Klotho knockdown. Furthermore, Klotho expression was upregulated by G-Rg1 treatment at the mRNA and protein levels. Our results suggest that G-Rg1 may be beneficial for ameliorating renal fibrosis by targeting Klotho/TGF-β1/Smad signaling in UUO rats.

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  • Ming Ji, Nina Xue, Fangfang Lai, Xiaoying Zhang, Sen Zhang, Yuchen Wan ...
    2018 Volume 41 Issue 4 Pages 592-596
    Published: April 01, 2018
    Released: April 01, 2018
    [Advance publication] Released: February 07, 2018
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    Psoriasis is a chronic inflammatory skin disease characterized by red, scaly and raised plaques. Thus far, T-cell infiltration is one of the most prominent pathogenic triggers, however, the exact molecular mechanisms underlying psoriasis have not been clearly established. Sphingolipid sphingosine-1-phosphate (S1P) is a lysophospholipid regulator modulating a variety of immune cell trafficking via interactions with its cognate receptors, S1P1–5. Activation of S1P signaling has recently emerged as a novel therapeutic avenue for psoriasis treatment. Here, we test a newly developed selective S1P1 modulator, Syl930, in four different psoriasis animal models. Our data reveals that oral administration of Syl930 can induce strong anti-proliferative and anti-inflammatory effects. Specifically, Syl930 decreases the pathological thickening of back skin induced by sodium lauryl sulfate (SLS), inhibits the proliferation of basal cells in a vaginal epithelium model and increases the granular layer scales in a mouse tail assay. Moreover, Syl930 can ameliorate the parakeratosis and acanthosis as well as improve granular layer composition and decrease the thickening of epidermis in a propranolol-induced guinea pig psoriasis model. Therefore, we demonstrate that Syl930 is a promising candidate for psoriasis therapy in clinical.

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  • Yukihiro Nomura, Hiroyuki Murata, Hiroaki Sasai, Akihiko Kimura, Takao ...
    2018 Volume 41 Issue 4 Pages 597-603
    Published: April 01, 2018
    Released: April 01, 2018
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    Unusual bile acids (1β-hydroxylated bile acids), particularly 1β-hydroxyl-cholic acid (CA-1β-ol) and 1β-hydroxyl-chenodeoxycholic acid (CDCA-1β-ol), have been detected in the urine of infants. These acids are conjugated with amino acids, such as taurine, and are then excreted mainly via the urine. CA-1β-ol and CDCA-1β-ol are the predominant bile acids during infancy and are present in relatively large amounts in the urine. However, the biosynthetic pathway of 1β-hydroxylated bile acids in infants remains unclear. To investigate the biosynthetic pathway of 1β-hydroxylated bile acids during infancy, we performed a metabolic reaction using infant hepatocytes at 3 months after delivery. Glyco- and tauro-CA-1β-ol were identified by LC/tandem mass spectrometry (MS/MS) analysis of the extracted culture medium incubated with cholic acids (CAs). Further, we identified that ketoconazole suppressed CA 1β-hydroxylation and that the CYP3A subfamily was the primary group of enzymes responsible for CA-1β-ol formation. The present study provides new information about the biosynthetic pathway of 1β-hydroxylated bile acids during infancy.

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  • Nobutaka Ogasawara, Tamami Kudo, Masaki Sato, Yasushi Kawasaki, Sei Yo ...
    2018 Volume 41 Issue 4 Pages 604-611
    Published: April 01, 2018
    Released: April 01, 2018
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    CRIM1 is a membrane protein that has been reported to be related to cell proliferation. CRIM1 is expressed in renal carcinoma cells, but its involvement in proliferation and malignant transformation remains unclear. We analyzed whether alterations in the characteristics of cancer cells are observed following knockdown of CRIM1. Decreased expression of CRIM1 did not affect proliferation or anchorage-independent growth. The results of wound healing and invasion assays showed that reduced expression of CRIM1 increased cells’ migratory and invasive abilities. Expression analysis of factors involved in migration and invasion in CRIM1-knockdown cells revealed that expression of the cell adhesion factor E-cadherin declined and expression of claudin-1, which is upregulated in metastatic cancer cells, increased. In addition, increased expression of matrix metalloproteinase (MMP) 2 and MMP9, protease essential for cancer cell invasiveness, was observed. Furthermore, an increase in phosphorylated focal adhesion kinase (FAK), which increases cell migration, was observed. Increased expression of the E-cadherin transcription repressors Snail, Slug, and ZEB-1 were observed, and mRNA levels of E-cadherin were decreased. Therefore, expression of E-cadherin is thought to be decreased by both suppression of E-cadherin mRNA expression and promotion of degradation of the E-cadherin protein. In addition, expression of CRIM1 was decreased in renal cancer cells undergoing epithelial–mesenchymal transition (EMT) stimulated by tumor necrosis factor alpha (TNF-α). Thus, CRIM1 regulates the expression of several EMT-related factors and appears to play a role in suppressing migration and invasion through control of EMT.

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  • Kumkum Rahman Mouree, Naoki Kishimoto, Nozomi Iga, Chie Kirihara, Keng ...
    2018 Volume 41 Issue 4 Pages 612-618
    Published: April 01, 2018
    Released: April 01, 2018
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    Human immunodeficiency virus type 1 (HIV-1) recruits diverse cellular factors into viral particles during its morphogenesis, which apparently play roles in modulating its infectivity. In our study, proteomic techniques demonstrated that a key glycolytic protein, pyruvate kinase muscle type 2 (PKM2), is incorporated into viral particles. Here, we show that virion-packaged PKM2 significantly reduces viral infectivity by affecting the incorporation level of a cellular tRNALys3 into virions. Enhanced expression of PKM2 in HIV-1-producing cells led to a higher incorporation level of PKM2 into progeny virions without affecting the viral maturation process. Compared with the control virus, the high-level-PKM2-packaging virus showed decreased levels of both reverse transcription products and cellular tRNALys3 packaging, suggesting that the shortage of intravirion tRNALys3 suppresses reverse transcription efficiency in target cells. Interestingly, the enhanced expression of PKM2 also suppressed the virion recruitment of other nonpriming cellular tRNAs such as tRNALys1,2 and tRNAAsn, which are known to be selectively packaged into virions, without affecting the steady level of the cytoplasmic pool of those tRNAs in producer cells, suggesting that PKM2 specifically impedes the selective incorporation of tRNAs into virions. Taken together, our findings indicate that PKM2 is a vital host factor that negatively affects HIV-1 infectivity by targeting the tRNALys3-mediated initiation of reverse transcription in target cells.

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    Editor’s picks

    Human immunodeficiency virus type 1 (HIV-1) recruits diverse cellular factors into viral particles during its morphogenesis, which apparently play roles in modulating its infectivity. The article by Mouree et al. evaluated that a key glycolytic protein, pyruvate kinase muscle type 2 (PKM2) is incorporated into viral particles. Furthermore, the virion-packaged PKM2 significantly reduces the viral infectivity by affecting the selective packaging of intravirion tRNALys3, which primes the initiation of reverse transcription, along with other nonpriming tRNAs, such as tRNALys1,2 and tRNAAsn, without affecting the cytoplasmic level of these tRNAs. These findings proposed that PKM2 is a vital host factor that negatively affects HIV-1 infectivity by targeting the tRNALys3-mediated initiation of reverse transcription in target cells.

  • Hiroto Hatakeyama, Takuya Fujiwara, Hiromi Sato, Ayu Terui, Akihiro Hi ...
    2018 Volume 41 Issue 4 Pages 619-627
    Published: April 01, 2018
    Released: April 01, 2018
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    Acquired resistance to sunitinib is a challenge in the treatment of renal cell carcinoma (RCC). The dysregulation of cellular metabolism is prevalent during resistance acquisition. It is known that in sunitinib-resistant RCC 786-O (786-O Res) cells sunitinib is mainly sequestered in the intracellular lysosomes. However, the relevance between sunitinib resistance and cellular metabolism has not been examined. In this study, we examined the metabolic changes in 786-O Res by using capillary electrophoresis-time of flight mass spectrometry. The cell line 786-O Res was established via persistent treatment with sunitinib, where increase in intracellular sunitinib, and sizes of lysosomes and nuclei were enhanced as compared with those in the parental 786-O (786-O Par) cells. Metabolic analyses revealed that out of the 110 metabolites examined, 13 were up-regulated and 4 were down-regulated in the 786-O Res cells. The glycolysis, tricarboxylic acid cycle and pentose phosphate pathway (PPP) were identified as being altered in the sunitinib-resistant cells, which resulted in the enhanced metabolisms of energy, nucleic acids, and glutathione redox cycle. As sunitinib was sequestered in the enlarged lysosomes in 786-O Res, the enriched energy metabolism might contribute to the maintenance of luminal pH in lysosomes via the H+ ATPase. The changes in the PPP could contribute to nuclei enlargement through up-regulation of nucleic acid biosynthesis and protect 786-O Res from cytotoxicity induced by sunitinib through up-regulation of reduced glutathione. Though the direct link between sunitinib resistance and metabolic alternation remains to be elucidated, this metabolomics study provides fundamental insights into acquisition of sunitinib resistance.

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  • Yuriko Wakita, Takashi Sato, Keisuke Chosa, Mary Ann Suico, Ryoko Sasa ...
    2018 Volume 41 Issue 4 Pages 628-636
    Published: April 01, 2018
    Released: April 01, 2018
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    Transthyretin (TTR) is a tetrameric beta-sheet-rich protein that is important in the plasma transport of thyroxine and retinol. Mutations in the TTR gene cause TTR tetramer protein to dissociate to monomer, which is the rate-limiting step in familial amyloid polyneuropathy. Amyloidogenicity of individual TTR variants depends on the types of mutation that induce significant changes in biophysical, biochemical and/or biological properties. G101S TTR variant was previously identified in a Japanese male without amyloidotic symptom, and was considered as a non-amyloidogenic TTR variant. However, little is known about G101S TTR. Here, we found slight but possibly important biophysical differences between wild-type (WT) and G101S TTR. G101S TTR had slower rate of tetramer dissociation and lower propensity for amyloid fibril formation, especially at mild low pH (4.2 and 4.5), and was likely to have strong hydrophobic interaction among TTR monomers, suggesting relatively higher stability of G101S TTR compared with WT TTR. Cycloheximide (CHX)-based assay in HEK293 cells revealed that intracellular G101S TTR expression level was lower, but extracellular expression was higher than WT TTR, implying enhanced secretion efficiency of G101S TTR protein compared with WT TTR. Moreover, we found that STT3B-dependent posttranslational N-glycosylation at N98 residue occurred in G101S TTR but not in other TTR variants, possibly due to amino acid alterations that increase N-glycosylation preference or accelerate rigid structure formation susceptible to N-glycosylation. Taken together, our study characterizes G101S TTR as a stable and N-glycosylable TTR, which may be linked to its non-amyloidogenic characteristic.

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  • Miku Kuritani, Nobuhiro Sakai, Akiko Karakawa, Motoki Isawa, Masahiro ...
    2018 Volume 41 Issue 4 Pages 637-643
    Published: April 01, 2018
    Released: April 01, 2018
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    Denosumab is an anti-bone resorptive drug consisting of complete human monoclonal antibodies that targets receptor activator of nuclear factor κB ligand (RANKL), which is responsible for osteoclast formation. The drug has been adapted for bone diseases, such as osteoporosis and bone metastasis related to cancer, but is not used for alveolar bone destruction related to periodontitis. In the present study, we aimed to clarify whether denosumab prevents bone destruction associated with lipopolysaccharide (LPS)-induced calvaria inflammation and experimental periodontitis in model mice. Denosumab does not bind to mouse RANKL, thus we used anti-mouse monoclonal RANKL antibodies. We also examined the inhibitory effects toward bone destruction of another anti-bone resorptive drug zoledronate, a nitrogen-containing bisphosphonate. Local administration of anti- RANKL antibodies into the calvaria area inhibited LPS-induced osteoclast formation and bone destruction, while zoledronate inhibited bone destruction but not osteoclast formation due to its different action mechanism. In periodontitis model mice, in which the second molars were ligated with a silk suture to induce inflammation, intraperitoneal administration of anti-RANKL antibodies significantly inhibited alveolar bone destruction and tooth root exposure. On the other hand, zoledronate only weakly repressed alveolar bone destruction and failed to inhibit root exposure. These results suggest that denosumab is a promising candidate to prevent alveolar bone destruction associated with periodontitis.

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Notes
  • Hiroaki Tanaka, Koichi Takahashi, Kazunori Yamaguchi, Keiichi Kontani, ...
    2018 Volume 41 Issue 4 Pages 644-648
    Published: April 01, 2018
    Released: April 01, 2018
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    Bevacizumab (BV), an inhibitor of vascular endothelial growth factor, is used in combination with paclitaxel (PTX) to treat advanced breast cancer. Hypertension and proteinuria are characteristic adverse events of BV therapy. We assessed the potential of these adverse events as predictors of BV treatment responses. Our results revealed that groups that developed hypertension and proteinuria early (by day 56) had a stronger antitumor response (Fisher’s exact test p<0.05). However, no significant difference was observed in progression-free survival (the Kaplan–Meier method and Log-rank test). As a reference, age, the treatment line, subtypes, liver and renal function, diabetes mellitus and hyperlipidemia history, body mass index, influencing concomitant medicine, average relative dose intensity and hematotoxicity did not significantly differ between groups with or without hypertension and with or without proteinuria. These results indicate the potential of the development of hypertension and proteinuria as predictors of improved outcomes with PTX plus BV therapy in patients with breast cancer. However, since both adverse events may preclude the continuation of treatment, their earlier management may be required.

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  • Hideya Kokubun, Chizuko Takigawa, Kanako Miyano, Yasuhito Uezono
    2018 Volume 41 Issue 4 Pages 649-651
    Published: April 01, 2018
    Released: April 01, 2018
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    In March 2013, the clinical use of oral methadone tablets was initiated in Japan. There are many factors responsible for the change in blood concentrations of methadone, and its pharmacokinetics is very complex. Therefore, a simple and accurate measurement method for methadone blood concentrations was developed using HPLC/electrochemical detector (ECD). An eluent of 10 mM Na2HPO4/CH3CN/CH3OH (20 : 19 : 3) was used as the mobile phase. The column was used the XTerra® RP18, and the voltage of the ECD was set at 400 to 800 mV. As a result, the calibration curve was linear in the ranges of 10 to 100 ng/mL (y=5012.7x+1041.1, r=0.999). The intra- and inter-day coefficients of variation were <5.2 and <5.8%, respectively. Therefore, this method was considered to be useful for the measurement of methadone blood levels in cancer patients. Also, using this method, blood methadone concentration was measured over time in a patient with cancer-associated pain who was treated with methadone. The estimated clearance (CL/F) and distribution volume (Vd/F) of methadone were 2.84 L/h and 502.8 L, respectively, and took about two weeks to reach steady state.

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  • Kosuke Shimizu, Satomi Yamaguchi Ikeuchi, Ai Okajima, Genki Nakamura, ...
    2018 Volume 41 Issue 4 Pages 652-656
    Published: April 01, 2018
    Released: April 01, 2018
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    The tumor microenvironment is one of the key factors contributing to the efficiency of drug delivery to a tumor. It has been reported that lymphangiogenesis is induced in certain tumors. Because the lymphatic system functions as a drainage one, it is possible that tumor lymphatic vessels alter not only the tumor microenvironment, but also the distribution of drug nanocarriers accumulated in the tumor tissue. Herein, we aimed to elucidate the involvement of the tumor lymphatic system in the translocation of intratumoral liposomes to regional lymph nodes by using vascular endothelial growth factor (VEGF)-C-overexpressing B16F10 tumor-bearing mice (B16/VEGF-C). When the amount of polyethylene glycol (PEG)-modified liposomes in lymph nodes (cervical, brachial, axillary, and inguinal lymph nodes) was measured after the radiolabeled liposomes had been intratumorally injected into B16/VEGF-C-bearing mice or wild-type B16-bearing mice, the accumulation of liposomes in the axillary and inguinal lymph nodes was significantly higher on the tumor-implanted side of B16/VEGF-C-bearing mice than on that of the B16-bearing ones. On the other hand, the accumulation of liposomes in these lymph nodes on the control side (no implantation) of either type of tumor-bearing mice was very low; and no difference could be observed between the 2 sides. Furthermore, the intratumoral distribution of liposomes was observed to be located near the lymphatic vessels. These results indicate that the tumor lymphatic system contributed to the extrusion of a portion of PEG-modified liposomes from the tumor tissue, suggesting that tumor lymphangiogenesis would be one of the key factors to determine the intratumoral distribution of liposomes and their subsequent fate.

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  • Kohei Sone, Asami Mori, Kenji Sakamoto, Tsutomu Nakahara
    2018 Volume 41 Issue 4 Pages 657-660
    Published: April 01, 2018
    Released: April 01, 2018
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    We previously reported that systemic administration with sodium hydrogen sulfide, a rapid-release donor compound of hydrogen sulfide (H2S), protected retinal neurons against N-methyl-D-aspartic acid (NMDA)-induced injury. For clinical application of H2S donors for retinal neurodegeneration, topical administration with an extended-release donor compound will be better. In the present study, we histologically investigated whether GYY4137, an extended-release hydrogen sulfide donor, had a protective effect on NMDA-induced retinal injury in the mice in vivo. Male and female B6.Cg-Tg(Thy1-CFP)23Jrs/J and C57BL/6J mice anesthetized with a mixture of ketamine and xylazine were subjected to intravitreal NMDA injection (80 nmol/eye). GYY4137 was intravitreally administered with NMDA simultaneously. Morphometric evaluation was carried out seven days after NMDA injection. Intravitreal NMDA induced retinal ganglion cell loss. GYY4137 (1, 10 and 100 nmol/eye) significantly reduced retinal ganglion cell loss seven days after NMDA injection. GYY4137 (10 nmol/eye) decreased the numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive and 8-hydroxy-2′-deoxyguanosine (8-OHdG)-positive cells 12 h after NMDA injection. These results suggest that extended release donor compounds of H2S protect retinal neurons against excitotoxicity induced by intravitreal NMDA in the mice in vivo through its anti-oxidative activity.

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