BPB Reports

2020 - Vol. 3

2020 - Vol. 3

Regular Article
Activation of TRPV4 Channel Regulates Differentiation to and Function of Myeloid-Derived Suppressor Cells Vol.3, No.2, p.70-75
Moeka Yamamoto , Ichiro Horie , Yoichiro Isohama , Mitsutoshi Tsukimoto
Received: December 23, 2019
Accepted: March 16, 2020
Released: March 25, 2020
Abstract Full Text PDF[2M]

Myeloid-derived suppressor cells (MDSCs), which are derived from immature bone marrow cell (BMC) populations that proliferate in the tumor microenvironment, suppress T cell immune responses. Transient receptor potential vanilloid (TRPV) 4, which is a Ca2+ channel, is involved in tumor growth, but the role of TRP channels in MDSC differentiation and function remains unclear. Here, we first investigated the involvement of TRP channels in the differentiation of MDSCs. The selective TRPV4 channel antagonist RN-1734 increased the population of MDSCs (CD11b+Gr-1+) at Day 3, while the TRPV4 agonist GSK1016790A decreased it, suggesting that stimulation of TRPV4 suppresses the differentiation of BMCs to MDSCs. GSK1016790A also increased the production of nitric oxide and reactive oxygen species, but suppressed the expression of Arg-1 mRNA, which encodes arginase-1, in MDSCs. Furthermore, GSK1016790A decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in MDSCs, thereby attenuating STAT3 signaling. Our results suggest that TRPV4 plays a role in regulating both the differentiation and function of MDSCs, and therefore could be a promising target for cancer immunotherapy.

Report
Interaction Between Piperacillin/Tazobactam and Warfarin: a Single-Center Retrospective Single-Arm Cohort Study Vol.3, No.2, p.65-69
Shota Kadomura , Yoh Takekuma , Shungo Imai , Hitoshi Kashiwagi , Kotaro Kawamoto , Tatsuya Itoh , Mitsuru Sugawara
Received: January 26, 2020
Accepted: March 09, 2020
Released: March 16, 2020
Abstract Full Text PDF[1011K]

Antibiotics influence the anticoagulation effect of warfarin and increase the bleeding risk in patients who are receiving warfarin. Piperacillin/tazobactam (PIPC/TAZ) is commonly used to treat infections such as healthcare-associated infection. However, there have been few reports about the interaction between warfarin and PIPC/TAZ. In this study, we investigated the influence of PIPC/TAZ on the anticoagulation effect of warfarin in hospitalized patients. The primary outcome was elevation of prothrombin time international normalized ratio (PT-INR) after PIPC/TAZ administration. Secondary outcomes were the proportion of patients with supratherapeutic levels of PT-INR, discontinuation of administration or reduction in the dose of warfarin, bleeding, transfusion, and vitamin-K rescue. Fifteen patients were enrolled in this study. PT-INR elevation occurred in 11 (73.3%) of the 15 patients. The median value of PT-INR after administration of PIPC/TAZ was significantly higher than the value before administration: 2.22 (interquartile range (IQR), 2.05-2.76) and 1.90 (IQR, 1.36-2.45), respectively (p = 0.024). Three (20%) of the 15 patients had PT-INR over 4, and discontinuation of administration or reduction in the dose of warfarin was needed in 6 (40%) of the 15 patients. Bleeding occurred in one patient, transfusion was performed in one patient and vitamin-K rescue was performed in one patient. This study showed that PIPC/TAZ induced elevation of PT-INR in patients receiving warfarin and that discontinuation or reduction in the dose of warfarin was needed in 40% of the patients. Therefore, we recommend to close monitoring of PT-INR in patients treated with warfarin during PIPC/TAZ administration.

Report
Urinary Bile Acid Shows Diurnal Fluctuation and Phase Shift with Daytime-Restricted Feeding in Rats Vol.3, No.2, p.60-64
Hiroshi Kawai , Ai Kurokawa , Takuya Ishibashi , Reiko Iwadate , Naomi Kudo , Yoichi Kawashima , Atsushi Mitsumoto
Received: January 09, 2020
Accepted: March 02, 2020
Released: March 16, 2020
Abstract Full Text PDF[804K]

Dysregulation of the biological clock disrupts the homeostasis of physiological functions, which may lead to the development of various disorders. To investigate the relationship between biological rhythms and disorders, an efficient monitoring method of the biological clock is necessary. In this study, we analyzed the circadian rhythmicity profile of bile acids in urine and investigated whether urinary bile acid (UBA) could reflect the circadian rhythm in liver physiology. Male Wistar-Hannover rats were maintained in metabolic cages under ad lib feeding and later subjected to restricted feeding (in which rats were fed only during light periods). Excreted urine was collected in each session, and bile acid contents were analyzed via an enzyme-based total bile acid assay. UBA content showed diurnal fluctuations under both ad lib and restricted feeding conditions and reached a peak during the dark period of ad lib feeding. In contrast, with restricted feeding, the peak was observed during light periods. Restricted feeding induced an 8-12 h phase shift. Diurnal fluctuation and phase shift by restricted feeding are distinctive features controlled by biological clocks in peripheral tissues. Since bile acids are synthesized in the liver, we propose that UBA might reflect the circadian fluctuation in liver physiology.

Report
(–)-Isostemonamine Can Enhance the Anti-Proliferative Activity of Trichostatin A Against Human Breast Cancer MDA-MB-231 Cells Vol.3, No.2, p.56-59
Masayo Hirao-Suzuki , Shuso Takeda , Takayuki Iwata , Satoshi Fujita , Taishi Tomiyama , Masufumi Takiguchi , Akihisa Toda , Mitsuru Shindo
Received: January 06, 2020
Accepted: February 13, 2020
Released: March 12, 2020
Abstract Full Text PDF[1M]

Recent findings established (–)-isostemonamine as an anti-proliferator for estrogen receptor α-negative human breast cancer cells, MDA-MB-231, known to grow/divide at an aggressive rate. However, ST-4, a thioamide derivative of (–)-isostemonamine, is believed to not affect the viability of these cells. Epigenetic changes, such as DNA methylation and histone modification, are involved in the progression of many cancers, including breast cancer. In the present study, we investigated whether ST-4 and its related compounds (ST-3 and ST-5) can potentiate the anti-proliferative activity of the established epigenetic modifiers, 5-aza-2’-deoxycytidine (5-aza-dC; a DNA methyltransferase 1 inhibitor), trichostatin A (TSA; a class I/II histone deacetylase inhibitor), and etoposide (a DNA topoisomerase IIα inhibitor). Data obtained from this study demonstrate that, among the studied compounds, ST-4 displays the strongest enhancement of the anti-proliferative activity of TSA, against MDA-MB-231 cells (IC20 of TSA = 14 ± 3.4 nM versus that of TSA/ST-4 combination = 7.8 ± 1.1 nM). However, this effect was not observed at higher concentrations of above 25 nM of TSA, at which the IC50 values of TSA with or without ST-4 were not significantly different (30 ± 4.4 nM versus 28 ± 1.4 nM, respectively). Results from the study suggest that combining ST-4 with established anti-cancer agents could potentiate the latter’s anti-proliferative activity, thereby potentially minimizing the concentration of these agents needed for optimal clinical efficacy and safety.

Regular Article
Involvement of TRPV1 and TRPV4 Channels in Enhancement of Metastatic Ability Induced by γ-Irradiation in Human Lung Cancer A549 Cells Vol.3, No.1, p.50-55
Kurumi Washiya , Rie Sasaki , Yuto Nakanishi , Kazuki Kitabatake , Keisuke Nishino , Sei-ichi Tanuma , Shuji Kojima , Mitsutoshi Tsukimoto
Received: January 17, 2020
Accepted: February 15, 2020
Released: February 27, 2020
Abstract Full Text PDF[4M]

Radiation therapy is an important local treatment for malignant tumors, but ionizing radiation may also facilitate tumor invasion and metastasis. The transient receptor potential (TRP) superfamily, which is a diverse group of ion channels activated by various stimuli, has a variety of pathophysiological functions, including a role in malignancy. However, it is not clear whether TRP channels influence radiation-induced biological effects. Here, we show that TRPV1 and TRPV4 channels contribute to the γ-irradiation-induced enhancement of migration of human lung cancer A549 and mouse melanoma B16 cells. We found that γ-irradiation induced both cell migration and actin stress fiber formation of A549 cells, but both effects were suppressed by the TRPV1 inhibitors capsazepine AMG9810, SB366791, and BCTC, and by the TRPV4 inhibitors RN-1734 and GSK2193874. γ-Irradiation induced migration was also suppressed by knockdown of TRPV1 and TRPV4 channels. Furthermore, γ-irradiation of B16 mouse melanoma cells increased the number of lung metastases in C57BL/6 mice, compared to non-irradiated B16 cells, and TRPV1 and TRPV4 channel inhibitors suppressed this increase. These results suggest that TRPV1 and TRPV4 channels are potential targets for intervention to block the acquisition of motility by cancer cells during radiotherapy in order to reduce the risk of metastasis.

Report
Effect of Remifentanil-Based Anesthesia on Perioperative Phagocytic Function of Human Monocytes Vol.3, No.1, p.45-49
Manzo Suzuki , Yoshinori Abe , Yusuke Taguchi , Hiroyasu Bito
Received: December 29, 2019
Accepted: February 10, 2020
Released: February 19, 2020
Abstract Full Text PDF[1M]

Although morphine-induced modulation of immune cells has been well studied, modulation of immune cells by fentanyl and remifentanil, the latter of which has been used in recent anesthesia procedures, has not been well-studied. Our aim was to identify the effects of fentanyl and remifentanil on phagocytosis and respiratory burst in leucocytes in in vivo and in vitro studies. In the in vivo study, twelve patients were assigned to receive fentanyl-based anesthesia (fentanyl group, n=6) or remifentanil-based anesthesia (remifentanil group, n=6). Blood samples were obtained from before anesthesia to 30 min after anesthesia in each group. In the in vitro study, blood samples were obtained from three healthy volunteers and incubated with various blood concentrations of fentanyl or remifentanil (from 0.3 ng/mL to 9 ng/mL). Phagocytic activity (percentage of phagocyting cells) and respiratory burst activity (percentage of cells producing oxygen radicals) were analyzed. In the in vivo study, phagocytosis was suppressed only before incision in the fentanyl group whereas suppression of phagocytosis was continued until emergence from anesthesia in the remifentanil group. In the in vitro study, incubation with fentanyl or remifentanil tended to enhance phagocytic function of monocytes and had no dose-dependent effect over various concentrations of fentanyl or remifentanil, respectively. Remifentanil-based anesthesia suppressed the phagocytic function of monocytes during its administration.

Regular Article
Persistence, Effects, and Adverse Events Associated with Real-World Daily Teriparatide Use in Japanese Patients with Osteoporosis Vol.3, No.1, p.39-44
Tomoko Matsumoto , Kazuyuki Niki , Asuka Hatabu , Takuya Oshima , Kimihiko Sato , Mikiko Ueda , Tetsuya Sato , Etsuko Uejima
Received: November 06, 2019
Accepted: January 06, 2020
Released: February 14, 2020
Abstract Full Text PDF[812K]

Few studies have addressed the effects and adverse events associated with daily teriparatide use, as well as the adherence and causes for non-adherence, among Japanese patients with osteoporosis, particularly those older than 80 years. In this study, we aimed to clarify various parameters associated with daily teriparatide use in Japanese patients with osteoporosis in a real-world clinical setting. This retrospective study compared the persistence of daily teriparatide use and the associated effects and adverse events in older (≥80 years, n=52) and younger patients (<80 years, n=106) treated with teriparatide between May 2013 and May 2018 at a single orthopedic clinic. We observed a significantly higher treatment completion rate among younger patients compared to their older counterparts (59.6% vs. 40.6%, p=0.036). Of the 74 patients (both patient groups) who completed a 24-month treatment course, only one (1.35%) developed new vertebral fractures. Our findings suggest that older patients would benefit from consistent osteoporosis treatment, particularly with a generally safe and effective agent, such as teriparatide. However, Log-rank test also shows the older patients exhibits a greater tendency to drop out than the younger patients (p=0.0238). The older patients tended to continue to drop out from the beginning. Accordingly, our results emphasize the importance of interventions, especially continuous encouraging from the first self-injection of teriparatide.

Regular Article
Cadmium Inhibits All-Trans-Retinoic Acid-Induced Increase of Nitroblue Tetrazolium Reduction Activity and Induces Metallothionein 1G Expression in Human Acute Myelocytic Leukemia HL-60 Cells Vol.3, No.1, p.34-38
Shoko Ogushi , Shuji Ikemoto , Nobuhiko Miura , Tsuyoshi Nakanishi , Tomoki Kimura
Received: January 07, 2020
Accepted: January 30, 2020
Released: February 10, 2020
Abstract Full Text PDF[2M]

Cadmium is an environmental pollutant. Metallothioneins are cysteine-rich, low-molecular-weight proteins that are induced by cadmium, which they chelate for detoxification. In humans, the functional metallothionein isoforms are MT1A, 1B, 1E, 1F, 1G, 1H, 1M, 1X, 2A, 3, and 4. It has been reported that overexpression of MT1G inhibits all-trans-retinoic acid (ATRA)–induced hematopoietic differentiation. Here, we found that cadmium inhibited ATRA-induced nitroblue tetrazolium reduction activity, a marker of hematopoietic differentiation, in human acute myelocytic leukemia HL-60 cells. Reverse transcription – quantitative polymerase chain reaction analysis of HL-60 cells revealed the expression of the metallothionein isoforms MT1G, MT1X, and MT2A and showed that MT1G expression increased significantly after cadmium treatment. ATRA treatment significantly attenuated this cadmium-induced increase in MT1G expression; however, MT1G expression remained significantly higher than that in untreated cells. No significant changes in MT1X and MT2A expression were observed. We also found that the cadmium-induced increase in MT1G expression was independent of CpG demethylation. ATRA is a standard chemotherapy treatment for acute promyelocytic leukemia; our findings suggest that cadmium may inhibit the effect of this chemotherapy.

Regular Article
Reconstitution of Bacterial Tyrosine Kinase-Modulator Interaction in a Human Cell Line Vol.3, No.1, p.28-33
Hidesuke Fukazawa , Mari Fukuyama , Yoshitsugu Miyazaki
Received: October 21, 2019
Accepted: January 30, 2020
Released: February 07, 2020
Abstract Full Text PDF[4M]

Many bacterial species express tyrosine kinases termed BY-kinases that share no homology with eukaryotic enzymes. We have previously reported that the Staphylococcus aureus BY-kinase CapB2 when fused with the C-terminal activation domain of its modulator CapA1, can translate into an active tyrosine kinase in HEK293T cells. In the present study, full-length CapA1 and CapB2 tagged with different fluorescent proteins were transfected into HEK293T cells. When expressed individually, the modulator CapA1, a membrane protein in bacteria, also appeared to localize to the cell membrane in HEK293T cells. In contrast, the catalytic subunit, CapB2, was found to be cytosolic. Coexpression of the two proteins resulted in apparent translocation of CapB2 to the membrane with concomitant activation of tyrosine kinase activity. This translocation and activation of CapB2 did not occur when the cytoplasmic C-terminal tail of CapA1 was deleted. Conversely, the CapA1 cytoplasmic C-terminal tail alone, when attached to a membrane localization sequence, was sufficient for CapB2 translocation and kinase activation. Our results indicate that the kinase activity of CapB2 is stimulated by direct interaction with the C-terminal cytoplasmic domain of CapA1 and that the process can be reconstituted and visualized in a human cell line. We created various mutants of CapA and CapB, and present data that demonstrate the correlation between CapA-CapB interaction and kinase activation.

Regular Article
Role of Metallothionein in Transcriptional Regulation by Metal-Responsive Element-Binding Transcription Factor 1 Vol.3, No.1, p.22-27
Yu-ki Tanaka , Yoshiaki Futami , Yasunori Fukumoto , Noriyuki Suzuki , Yasumitsu Ogra
Received: November 15, 2019
Accepted: December 16, 2019
Released: February 05, 2020
Abstract Full Text PDF[1M]

Owing to the high metal binding affinity of metallothionein (MT), newly synthesized MT was speculated to attenuate the activity of metal-responsive element (MRE)-binding transcriptional factor 1 (MTF-1) by removing Zn from the activated MTF-1. To investigate the potential role of MT in the inactivation of MTF-1, we examined the transcriptional levels of reporter and endogenous MRE-dependent genes using mouse embryonic fibroblasts (MEFs) established from MT-knockout (KO) and wild-type (WT) mice. The activation of MTF-1 by the Cd exposure of MT-KO MEFs was sustained for 12 h, whereas that of MT-WT MEFs showed the rapid attenuation. Consequently, MT was found to negatively regulate MTF-1 activity, which can control the expression of MT itself.

Regular Article
The Antibiotic Cefotaxime Works as Both an Activator of Nrf2 and an Inducer of HSP70 in Mammalian Cells Vol.3, No.1, p.16-21
Mayuka Yamada , Midori Suzuki , Takuya Noguchi , Takumi Yokosawa , Yuto Sekiguchi , Natsumi Mutoh , Takashi Toyama , Yusuke Hirata , Gi-Wook Hwang , Atsushi Matsuzawa
Received: November 27, 2019
Accepted: January 23, 2020
Released: January 31, 2020
Abstract Full Text PDF[1M]

Both NF-E2-related factor 2 (Nrf2) and heat shock protein 70 (HSP70) contribute to cellular defense to various stresses, and have emerged as candidates of therapeutic targets to improve or prevent tissue damage. Cefotaxime (CTX), a third-generation cephalosporin antibiotic, is conceived as a safe drug largely free from side effects. CTX exhibits broad-spectrum antimicrobial activity, and thereby, is most commonly prescribed for the treatment of infectious diseases induced by Gram-positive or Gram-negative bacteria. In this study, we unexpectedly found the beneficial properties of CTX that upregulate both Nrf2 and HSP70 to the extent that stress-induced damage is ameliorated. Non-toxic levels of reactive oxygen species (ROS) induced by CTX activated the Nrf2 pathway without cytotoxicity, which in turn upregulated HSP70. Interestingly, the cytotoxicity of Fas/CD95 ligand (FasL), a cytotoxic cytokine that strongly induces apoptosis, was significantly ameliorated by pre-treatment with CTX, most likely because of the upregulation of Nrf2 and HSP70. Our results therefore show novel properties of CTX, which raise the possibility that CTX works as a non-toxic therapeutic agent for preventing and repairing tissue damage.

Regular Article
In Vitro Evaluation of the Interaction Between Activated Charcoal and N-Acetylcysteine after Acetaminophen Adsorption Vol.3, No.1, p.11-15
Yoshinori Tomoda , Mariko Fukumoto
Received: October 17, 2019
Accepted: January 11, 2020
Released: January 29, 2020
Abstract Full Text PDF[1M]

Gastrointestinal decontamination by activated charcoal (AC) is the most important treatment for acetaminophen (APAP) overdose. Because AC adsorbs a wide variety of toxins, it may also adsorb the oral antidote, N-acetylcysteine (NAC). NAC is a specific antidote for APAP overdose and administered as a 72-h oral regimen. We evaluated AC adsorption of NAC after APAP adsorption in vitro. Different concentrations of NAC solution diluted with simulated gastric fluids (SGF) and simulated intestinal fluids (SIF) were added to AC and incubated at 37°C for 1 h. The AC was then removed by filtration, and the NAC concentration was determined. This revealed that NAC was not only adsorbed onto the AC but also converted to N,N’-diacetyl-L-cystine (DAC), which is oxidized NAC. We then calculated the maximum adsorption capacity per gram of AC (Qm). The apparent Qm based on the amount of decreased NAC in the SGF was 400 mg/g, and that in the SIF was 714 mg/g. The actual Qm based on only the amount of adsorption in the SGF was 294 mg/g, and that in the SIF was 59 mg/g. We also determined whether or not AC could adsorb the loading and maintenance doses of NAC after APAP adsorption. The residual rate in the SGF was 2.1%, and that in the SIF was 0.3%. The rate of conversion to DAC was higher in the SIF than that in the SGF. By both the actions of adsorption and oxidation, AC may reduce the effect of loading and maintenance doses of NAC.

Regular Article
Construction of a PPARα Reporter Assay System with Drug-Metabolizing Capability Vol.3, No.1, p.7-10
Takuomi Hosaka , Ayano Wakatsuki , Takamitsu Sasaki , Ryota Shizu , Kouichi Yoshinari
Received: November 14, 2019
Accepted: December 29, 2019
Released: January 17, 2020
Abstract Full Text PDF[1M]

Peroxisome proliferator-activated receptor α (PPARα) belongs to the nuclear receptor superfamily and exerts hypolipidemic and anti-inflammatory functions when activated by ligand-binding. To screen its ligands, cell-based reporter assays have been widely used, but it is difficult to investigate the effects of the metabolites of test compounds on PPARα due to very low drug-metabolizing capability of cell lines generally used in those assays. The aim of this study was to construct a convenient PPARα reporter assay system with drug-metabolizing capability by using 9,000 x g supernatant (S9) of rat liver homogenate, which abundantly includes various drug-metabolizing enzymes. We used clofibrate as a model compound since it requires hydrolysis to clofibric acid to activate PPARα. In cell-based reporter assays using a PPARα-responsive luciferase reporter plasmid and a rat PPARα expression plasmid, reporter activity was increased by treatment with bezafibrate and clofibric acid, which directly activate PPARα as ligands, but not with clofibrate. The addition of S9 to culture media increased reporter activity of the cells treated with clofibrate, as expected. When heat-denatured S9 was used or a carboxylesterase inhibitor was included in the system, clofibrate-induced PPARα activation was not observed, suggesting that carboxylesterases are responsible for the hydrolysis of clofibrate to clofibric acid. Taken together, we have established a convenient PPARα reporter assay system with drug-metabolizing capability to assess PPARα-activating potency of both test compounds and their metabolites.

Regular Article
Factorial Analysis of Clostridioides Difficile Colitis and Pseudomembranous Colitis Using JADER Vol.3, No.1, p.1-6
Risako Takaya , Kana Misawa , Sho Tashiro , Yuki Enoki , Kazuaki Taguchi , Kazuaki Matsumoto
Received: October 25, 2019
Accepted: January 07, 2020
Released: January 15, 2020
Abstract Full Text PDF[1M]

Clostridioides difficile (C. difficile) colitis and pseudomembranous colitis are known as healthcare-associated intestinal infections. In this study, the incidence of C. difficile colitis and pseudomembranous colitis was investigated using the Japanese Adverse Drug Event Report (JADER). Using JADER data between April 2004 and September 2017, the patient who developed C. difficile colitis and pseudomembranous colitis were investigated. During the study period, 375 cases of C. difficile colitis and 903 cases of pseudomembranous colitis were reported. The numbers of reported cases of both C. difficile colitis and pseudomembranous colitis were largest in those in their 70s, accounting for 24.7% and 25.6%, respectively. Patients in their 60s-90s comprised the majority of all patients with both C. difficile colitis and pseudomembranous colitis. Both C. difficile colitis and pseudomembranous colitis were caused by antibiotics in many patients, and signals of all antibiotics were detected. In C. difficile colitis, signals of immunosuppressants, corticosteroids, and alkylating drugs were also detected among drugs other than antibiotics. For pseudomembranous colitis, the use of molecularly targeted drugs, antimetabolic drugs, and corticosteroids was reported other than antibiotics. Using JADER, we revealed risk factors for the development of C. difficile colitis and pseudomembranous colitis, and firstly revealed that molecularly targeted drugs other than antibiotics could also be potential risk factors. Our findings may be useful for the early detection of drug-induced C. difficile colitis and pseudomembranous colitis.