Publisher：Japanese Pharmacological Society  

<p>&lt;purpose&gt;</p><p>Hydrogen peroxide is known as one of ROS which gives oxidative stress to cells and induces apoptosis. However, details of the mechanism for cancer cells by hydrogen peroxide is still unknown.We have established "hydrogen peroxide resistant (HR) cancer cells" that are resistant to high concentration hydrogen peroxide. The mechanism of resistance to hydrogen peroxide acquired by HR cancer cells has not yet been elucidated, however if the mechanism becomes clear, it could be applied to cancer treatment. In this study, therefore, we aimed to elucidate its hydrogen peroxide resistance mechanism and carried out the following experiment.</p><p>&lt;method&gt;</p><p>Cell lines that continued to survive against graded hydrogen peroxide treatment of HeLa (up to 70 μM) and SAS (up to 35 μM) were subjected to hydrogen peroxide at the concentrations of 0, 25, 50, 75 and 100 μM respectively, and the cell viability was examined by WST assay. Subsequently, the endogenous catalase enzymatic activity of HR cancer cells was measured using Catalase Assey Kit (SIGMA).Furthermore, lipid peroxidation of HR cancer cells was analyzed by immunofluorescence using 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LOX) antibody. HNE is typical lipid peroxidation marker and 5-LOX is known as lipid peroxidase. </p><p>&lt;results and discussion&gt;</p><p>In HeLa and SAS parental cells, they survived to the extent of 25 μM by hydrogen peroxide treatment. On the other hand, stepwise hydrogen peroxide-treated cells survived up to100 μM (HeLa) and 50 μM (SAS), showingresistance to hydrogen peroxide. Analysis of catalase enzyme activity showed significant increase in HeLa HR cells compared with the HeLa parent, but there were no significant differences in SAS cells.</p><p>Furthermore, HNE and 5-LOX expression levels in HR cells were significantly decreased compared with the parental cells byimmunofluorescent staining. </p><p>As mentioned above, the membrane lipid peroxidation is regulated by the expression of 5-LOXrather than a catalase activity. The decreased 5-LOX expression may suppress lipids peroxidation in plasma membrane and leadto retention viability to hydrogen peroxide in HR cells.</p>

DOI： 10.1254/jpssuppl.92.0_2-P-122

Publisher：The Japanese Society of Conservative Dentistry  

<p> Purpose: Calcium hydroxide preparations and mineral trioxide aggregate (MTA) are used as pulp-capping materials. However, these materials do not adhere to teeth and dental restorations. The purpose of this study was to develop a new adhesive pulp-capping material.</p><p> Methods: In this study, the new pulp-capping material (PCX-TBB, Sun Medical, Tokyo, Japan) combined with 2-hydroxypropyl methacrylate and Portland cement, TheraCal LC (BISCO Dental Products, Schaumburg, IL, USA) and Dycal (Dentsply Sirona, York, PA, USA) were used. Discs of each material were immersed in distilled water, and the pH and calcium ion concentration were measured while exchanging water every 24, 48 and 72 hours. Next, human dental pulp-derived cells (DPCs: Dental Pulp Cells, AD010-F-RA, DV Biologics) were cultured with the discs in a 24-well plate. Then, the number of cells was measured after 24, 48 and 72 hours of culture. In addition, the dentin surfaces of 30 fresh bovine mandibular anterior teeth were used as the adhesion surface, and half of the dentin specimens were used as demineralized dentin. We cured each pulp-capping material on dentin, then performed shear bond strength tests on the bonded specimens.</p><p> Results: The pH was over 10.5 in distilled water for all the pulp-capping materials. In addition, PCX-TBB and TheraCal LC showed significantly higher cumulative calcium ion release values than Dycal at all times, and after 72 hours PCX-TBB showed the highest value. As for the effect on cell proliferation, each pulp-capping material group showed a significantly lower cell number than the control group; the PCX-TBB group was second after the control group at 72 hours, and the cell numbers of the TheraCal LC and Dycal groups were significantly lower.</p><p> PCX-TBB showed significantly higher shear bond strength than other materials under normal and demineralized dentin conditions. In addition, PCX-TBB and TheraCal LC showed significantly higher values for demineralized dentin than normal dentin. On the other hand, Dycal showed lower values for normal dentin and demineralized dentin.</p><p> Conclusions: In this study, PCX-TBB had a pH as high as that of currently marketed pulp-capping materials, had a sustained release of calcium greater than that of current products, and exhibited adhesion to teeth. PCX-TBB was shown to be an adhesive pulp-capping material.</p>

DOI： 10.11471/shikahozon.62.208

Publisher：Japanese Pharmacological Society  

<p>Although radiation therapy is one of the choices to treat cancers and is an excellent local treatment method, existence of radiation resistant cell is a major problem. We established clinically relevant radioresistant (CRR) cells that can survive exposing to 2 Gy/day X-rays for more than 30 days. However, the mechanism to obtain resistance has not been elucidated yet. We investigated the relationships between resistant mechanism to hydrogen peroxide (H₂O₂) and plasma membrane state in CRR cells. </p><p>　CRR cells showed resistance to H₂O₂, but catalase enzyme activity was down-regulated. Plasma membrane potential were low, no internal H₂O₂increase and no lipid peroxidation were seen even after 2 hours of H₂O₂treatment in CRR cells. Administration of oxidized lipid led to further cell death after H₂O₂ treatment in CRR cells. The lipoxygenase (ALOX) gene and protein expressions were down-regulated in CRR cells. We also established stress-sensitive ρ⁰ cells that lack mitochondrial DNA. Gene and protein expressions of ALOX were up-regulated in ρ⁰ cells. Expression of cyclooxygenase-2 does not seem to be involved in this mechanism. </p><p>　These results suggest that the involvement of ALOX in resistance or sensitivity of cancer treatment.</p>

DOI： 10.1254/jpssuppl.92.0_2-O-12

DOI： 10.1016/j.dib.2018.08.025

PubMed

Publisher：Tumor Biology  

DOI： 10.1177/1010428318799250

Scopus

PubMed

Publisher：公益社団法人 日本薬理学会  

<p>Mitochondria have been recognized as ¨Powerhouse of the Cell¨ for a long time. Lately, mitochondria have also been shown as the primary site of reactive oxygen species (ROS) generation and to be involved in apoptosis. Mitochondria have its own DNA (mtDNA) and mtDNA encodes 13 mitochondrial structure protein.It has been shown that mtDNA damage by ROS causes various types of diseases such as cancer. Therefore, these damaged cells will provide valuable cell models to study oxidative stress and overcome ROS derived diseases. We established mtDNA depleted ρ⁰ cells from HeLa and SAS cell lines and investigated the effect of ROS, especially hydrogen peroxide (H₂O₂) sensitivity. First, we performed H₂O₂ treatment on HeLa and SAS ρ⁰ cells at a concentration of 0 to 100 μM. Next, we investigated catalase gene expression and catalase enzyme activity. As a result, ρ⁰ cells showed high sensitivity to H₂O₂ compared with its parental cells, even though the catalase activity of ρ⁰ cells were up-regulated. We investigated cell membrane potential by DiBAC4 (3), lipid peroxidation by HNE immunostaining, endogenous H₂O₂ amount by HYDROP and uptake of H₂O₂ by stable isotope-containing H₂O₂. The result showed that the membrane potential of ρ⁰ cells was lower than their parental cells, the amount of HNE was elevated in ρ⁰ cells as compared with their parental cells, internal H₂O₂ amount was significantly increased only in ρ⁰ cells after 50 μM H₂O₂ treatment for 1 h and uptake of H₂O₂ were increased in ρ⁰ cells as compared with their parental cells. These changes in the membrane state, particularly lipid peroxidation, occurred in ρ⁰ cells as compared with their parental cells, leads to increase membrane permeability of H₂O₂. And the timing of the intracellular H₂O₂ concentration will be advanced. In conclusion, ρ⁰ cells are considered to be more susceptible to H₂O₂ than their parental cells because the membrane statuses change.</p>

DOI： 10.1254/jpssuppl.WCP2018.0_PO4-6-14

Publisher：公益社団法人 日本薬理学会  

<p>Early life stress leads to neurodevelopmental disorders. The excitatory-to-inhibitory functional switch of γ-aminobutyric acid (GABA switch) normally occurs on the up-regulation of K⁺-Cl^- cotransporter-2 (KCC2) in the first to the second postnatal week. GABA switch is necessary for normal construction of neural circuit. Therefore, early life stress may induce behavioral abnormalities through the influence of GABA switch. </p><p>　The purpose of this study is to examine the effect of maternal separation (MS) on the timing of GABA switch and on the adolescent behavior. </p><p>　Neonatal C57BL/6J male mice were divided into control group (Ctl) and experimental group. Experimental (MS) group was subjected to maternal separation for 3 hours a day during postnatal day 1 (P1) to P21. Then both groups were weaned at P22. We conducted quantification of KCC2 immunoreactivity (IR) at P7, 14, 21 and 35, and analyzed the timing of GABA switch by Ca²⁺ imaging method during P2 to P15. Behavioral analyzes such as open field, novel object recognition, elevated cross maze, attack action tests, were conducted from P35 to P38. MS resulted in a decrease in perimembrane KCC2-IR in the CA1 and CA3 regions at P7, 14, 21 and 35. NKCC1-IR in CA1 and CA3 were no significant difference between the Ctl and MS groups. Next, we analyzed the timing of GABA switch by Ca²⁺ imaging method. 10 μM of muscimol induced Ca²⁺ responses present in a higher percentage of neurons in the MS than in the Ctl on P8 and P11. The Ca²⁺ responses induced by muscimol were completely inhibited by pre-treatment with 10 μM of bicuculline. Compared to mice in the Ctl group on P35, MS group had decreased amounts of perimembrane KCC2 in hippocampal CA1 and CA3. There were no significant differences in NKCC1 in both groups on P35. We also found cognition and attention defects in MS group by behavioral analyzes. </p><p>　Taken together, neonatal MS inhibited the expression of perimembrane KCC2 for a long lasting period, leading to the delay timing of GABA switch in the hippocampus. The disrupted KCC2 expression could be associated with abnormal behaviors during adolescence.</p>

DOI： 10.1254/jpssuppl.WCP2018.0_PO3-1-49

Publisher：公益社団法人 日本薬理学会  

<p>Radiation therapy is one of the choices to treat malignant tumors. Although radiation therapy has been established as an excellent local treatment for malignant tumors, existence of radiation resistant cell is a major problem to overcome. To reveal radioresistant mechanism, we investigated using clinically relevant radioresistant (CRR) cells that had been obtained by exposing to 2 Gy/day X-rays for more than 30 days. CRR cells show not only radioresistance but also resistant to anticancer drug docetaxel. CRR cells also show low frequency of DNA double strand breaks, low mitochondrial membrane potential and produce little amount of reactive oxygen species (ROS). However, the molecular mechanism to obtain radio-resistant is not clear. So, we try to reveal the resistant ability to other oxidative stress such as hydrogen peroxide (H₂O₂). First, we investigated the resistance of CRR cells against H₂O₂ using WST assay. As a result, these cells showed resistant to H₂O₂. Next, we examined gene expression and enzyme activity of catalase that is H₂O₂ catabolic enzyme. Catalase expression was up-regulated in CRR cells. However, catalase enzyme activity was down-regulated. We also investigated mitochondrial DNA (mtDNA) copy number. It was shown that the mtDNA copy number was decreased in CRR cells. In addition, the amount of ATP, ATPase gene expressions and plasma membrane potential were investigated in CRR cells. It was shown that the amount of ATP decreased, ATPase gene expressions were up regulated and plasma membrane potential were low in CRR cells. Furthermore, increase of internal H₂O₂ amount and lipid peroxidation was investigated. As a result, increase of internal H₂O₂ amount and lipid peroxidation were seen in parental cells 2h after H₂O₂ administration. On the other hand, increase of internal H₂O₂ amount and lipid peroxidation were not seen in CRR cells 2h after H₂O₂ administration. These results suggest that the decrease of cell response through plasma membrane component is the main factor rather than internal oxidative stress scavenging enzyme activity to obtain resistance against oxidative stress in CRR cells.</p>

DOI： 10.1254/jpssuppl.WCP2018.0_PO3-7-32

Publisher：Biochemical and Biophysical Research Communications  

DOI： 10.1016/j.bbrc.2017.06.044

Scopus

PubMed

Publisher：Japanese Association of Regenerative Dentistry  

<p>It is advantageous to use adhesive root canal sealer for prevention of reinfection in pulpectomy. Mineral trioxide aggregate (MTA) is a hydraulic cement with bactericidal action and hard tissue induction ability and it has been widely used in dental treatment. The purpose of this study was to examine the effect of cell growth on adhesive root canal sealer containing white portland cement (WPC) for development of new adhesive root canal sealer with efficacy of MTA. There was no change in the number of cells in which human periodontal ligament cells survived in the culture medium containing components eluted from the experimental sealer in case of low concentration. The pH of the culture medium and the concentration of Calcium dissolved in the culture medium were WPC concentration-dependent increase. More research is required to determine if MTA-containing adhesive root canal sealer will induce hard tissue at root apex.</p>

DOI： 10.11223/jard.15.3

Event date： 2020.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：大阪　（誌上開催）  

Event date： 2019.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島  

Event date： 2019.11

Language：Japanese   Presentation type：Poster presentation  

Venue：沖縄  

Event date： 2019.11

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Venue：沖縄  

Event date： 2019.10

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Venue：東京  

Event date： 2019.9

Language：Japanese   Presentation type：Poster presentation  

Venue：静岡  

Event date： 2019.3

Language：Japanese   Presentation type：Poster presentation  

Venue：大阪  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：大阪  

Event date： 2018.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：大阪  

Event date： 2018.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡  

Event date： 2018.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡  

Event date： 2018.11

Language：Japanese   Presentation type：Poster presentation  

Venue：京都  

Event date： 2018.11

Language：Japanese   Presentation type：Poster presentation  

Venue：京都  

Event date： 2018.9

Language：English   Presentation type：Poster presentation  

Venue：福岡  

Event date： 2018.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡  

Event date： 2018.8

Language：Japanese   Presentation type：Poster presentation  

Venue：東京  

Event date： 2018.7

Language：English   Presentation type：Poster presentation  

Venue：京都  

Event date： 2018.7

Language：English   Presentation type：Poster presentation  

Venue：京都  

Event date： 2018.7

Language：English   Presentation type：Poster presentation  

Venue：京都  

Event date： 2018.2

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島