Japanese Center for Anti Aging MedSciences

Shōbara, Japan

Japanese Center for Anti Aging MedSciences

Shōbara, Japan
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Kato S.,Prefectural University of Hiroshima | Kato S.,Japanese Center for Anti Aging MedSciences | Saitoh Y.,Prefectural University of Hiroshima | Miwa N.,Prefectural University of Hiroshima | Miwa N.,Japanese Center for Anti Aging MedSciences
International Journal of Hyperthermia | Year: 2013

Purpose: The aim of this study was to evaluate inhibitory effects of L-ascorbic acid-2-O-phosphate-Na2 (APS), a pro-vitamin C, combined with hyperthermia on adipogenic differentiation of mouse stromal cells, OP9. Materials and methods: OP9 preadipocytes were differentiated with serum replacement, administered with APS, and simultaneously treated with hyperthermia using a capacitive-resistive electric transfer (CRet) apparatus, which was conducted repeatedly twice a day. After 2 days, intracellular lipid droplets were stained with Oil Red O, then observed by microscopy and assessed spectrophotometrically. Results: After stimulation by serum replacement for 2 days, lipid droplets were accumulated surrounding nucleus of OP9 cells. When APS of 0.15-0.6mM was administered without hyperthermia, the amount of lipid droplets was markedly suppressed to 50.5%∼-11.3% versus the undifferentiated control, and diminished huge aggregates of lipid droplets. In OP9 cells treated by hyperthermia at 42°C for 0.5min, 1min or 3min in the absence of APS, adipogenesis was suppressed abruptly in a time-dependent manner to 95.4%, 18.7% or -5.5%, respectively. Whereas, the percentage of adipogenesis was 96.8% in OP9 cells treated by mild hyperthermia alone at 41°C for 1min. The simultaneous application of APS and hyperthermia at 41°C for 1min markedly suppressed the accumulation of lipid droplets to 25.7%∼-66.2%. By scanning electron microscopy (SEM) observation, the surface of OP9 cells treated with APS and hyperthermia appeared to have the morphological property of undifferentiated OP9 cells. Conclusion: Combined treatment of APS and mild hyperthermia suppresses adipogenesis in OP9 cells, particularly in lipid droplets accumulation during spontaneous differentiation of OP9 preadipocytes. © 2013 Informa UK Ltd.


Kato S.,Osaka City University | Kato S.,Mie University | Kimura M.,Osaka City University | Miwa N.,Japanese Center for Anti Aging MedSciences
Journal of Biomedical Nanotechnology | Year: 2014

The synthetic thymidine analogue, 5-bromo-2′-deoxy-uridine (BrdU) was encapsulated in cationic liposome composed of dipalmitoylphosphatidylcholine, cholesterol and stearylamine (molar ratio = 1/1/0.2; diameter = 120 nm), and the radiosensitization of cationic liposomal BrdU was assessed on human melanoma cells HMV-II, with comparing to anionic or nonionic liposomal BrdU and free-BrdU. HMV-II cells were pretreated by cationic liposomal BrdU or free-BrdU and then exposed to X-ray, followed by WST-8 assay to examine cell proliferation. The radiation-induced change of nuclei was defined with Hoechst33342 staining. The rates of thymidine replacement by BrdU and DNA double-strand breaks on HMV-II cells were determined with an anti-BrdU antibody and anti-53BP1 antibody, respectively. On 7th day after X-ray irradiation at 3 Gy, cell proliferation was suppressed more markedly in the administration of cationic liposomal BrdU than free-BrdU at equivalent BrdU doses. Giant polykaryocytes were observed in cationic liposomal BrdU-treated HMV-II cells. Radiosensitization was enhanced dose-dependently along with BrdU doses of 0.1-0.8 μM in the order: cationic liposomal BrdU > anionic liposomal BrdU > nonionic liposomal BrdU ≒ free-BrdU. Similarly, the cationic liposomal BrdU augmented the rate of thymidine-moiety replacement by BrdU and DNA double-strand breaks more appreciably than free-BrdU. Therefore, the cationic liposome-encapsulation of BrdU would be one of favorable drug deliveries for facilitating the X-ray therapy against cancer. Copyright © 2014 American Scientific Publishers All rights reserved.


Kato S.,Osaka City University | Kato S.,Japanese Center for Anti Aging MedSciences | Kimura M.,Osaka City University | Kageyama K.,Osaka Butsuryo College | And 2 more authors.
Journal of Nanoscience and Nanotechnology | Year: 2012

The nitroimidazole-related hypoxic radiosensitizer, pimonidazole (Pmz) was encapsulated in liposome composed of dipalmitoylphosphatidylcholine, cholesterol and dipalmitoylphosphatidylglycerol (molar ratio = 1:1:0.2; diameter = 1129 nm), and the radiosensitization was evaluated in human melanoma cells HMV-II. Cell proliferation was examined by WST-8 assay after X-ray irradiation in the presence of liposomal Pmz or free-Pmz under hypoxic conditions. On 7th day after X-ray irradiation of 5 Gy, cell proliferation decreased more markedly in the administration of liposomal Pmz than free-Pmz at equivalent Pmz doses. Chromatin fragmentation or nuclear condensation was observed in liposomal Pmz-treated HMV-II cells. Radiosensitization was enhanced dose-dependently along with Pmz amounts of 250-2000 μM contained in liposomal Pmz. Intracellular uptake was more abundant for liposomal Pmz for 60-240 min than for free-Pmz. Thus liposomal Pmz has a potential to overcome radiation resistance in hypoxia, owing to enhanced intracellular uptake by melanoma cells. Copyright © 2012 American Scientific Publishers All rights reserved.


Kato S.,Prefectural University of Hiroshima | Kato S.,Mie University | Aoshima H.,Vitamin C60 BioResearch Corporation | Saitoh Y.,Prefectural University of Hiroshima | And 2 more authors.
Journal of Nanoscience and Nanotechnology | Year: 2014

Microcorpuscular titanium dioxide (TiO2), a useful sunscreen agent, photocatalyzes generation of reactive oxygen species (ROS). We assessed protective effects of fullerene-C60 derivatives or microcolloidal platinum (Pt) against ultraviolet ray (UV)-irradiation in the presence of TiO2 in vitro. UV-irradiation (8 J/cm2, mixed UVA and UVB) in the presence of 15 ppm TiO2 on HaCaT keratinocytes decreased cell viability as quantified by WST-1 assay, and increased both intracellular ROS and cell-membrane-lipid peroxidation, as quantified by nitroblue-tetrazolium (NBT) assay and diphenyl-1-pyrenylphosphine (DPPP) assay, respectively, whereas all of three phototoxicityrelated symptoms were appreciably repressed almost to UV-unirradiational levels by pretreatment with polyvinylpyrrolidone-entrapped fullerene-C60 (C60/PVP) or fullerene-C60 dissolved in squalane (C60/Sqn) in a dose-dependent manner of C 60, but scarcely by PVP alone or Sqn alone. In contrast, Pt repressed intracellular ROS generation, but did not prevent either peroxidation of cell-membranelipid or cell mortality. Then in the epidermis of 3-dimensional human skin tissue model, UV-irradiation in the presence of TiO2 extensively induced two symptoms such as ROS-generation around perinuclear regions and membrane-lipid peroxidation, both of which were repressed by C 60/PVP or C60/Sqn, whereas Pt did not prevent membrane-lipid peroxidation adequately. Thus the advantageous application of the lipophilic antioxidant fullerene-C60 which effectively protects cell membrane against peroxidation. In conclusion, fullerene-C60 can be expected to serve as an antioxidant for scavenging of TiO2- photocatalyzed ROS in the skin surface, and therefore provide a functional improvement of TiO2-containing sunscreens. Copyright © 2014 American Scientific Publishers.


Kato S.,Prefectural University of Hiroshima | Kato S.,Japanese Center for Anti Aging MedSciences | Saitoh Y.,Prefectural University of Hiroshima | Miwa N.,Prefectural University of Hiroshima | Miwa N.,Japanese Center for Anti Aging MedSciences
Journal of Nanoscience and Nanotechnology | Year: 2013

We investigated the anti-melanogenetic efficacy of hydrogen-occluding silica microcluster (H2-Silica), which is a silsesquioxane-based compound with hydrogen interstitially embedded in a matrix of caged silica, against melanogenesis in HMV-II human melanoma cells and L-DOPA-tyrosinase reaction [EC1.14.18.1]. HMV-II cells were subjected to oxidative stress by ultraviolet ray-A (UVA) exposure of 3-times of 0.65 J/cm2 summed up to 1.95 J/cm2. After UVA irradiation, HMV-II cells were stimulated to produce melanin by 2.72-fold more abundantly than unirradiated control. When HMV-II cells were treated with H2-Silica of 20 ppm or kojic acid of 28.4 ppm before and after UVA-irradiation, the amount of melanin was repressed to 12.2% or 14.5% as compared to that of UVA-irradiated control, respectively. That is, H2-Silica exhibited a comparable efficacy to the whitening agent kojic acid. The H2-Silica could prevent melanogenesis in HMV-II cells by low-level doses at 1-10 ppm, and cell viability and apoptosis event did not change even by high-level doses at 100-1000 ppm. On the contrary, kojic acid was cytotoxic at the concentration of 14-28 ppm or more. By microscopic observation, H2-Silica suppressed such properties indicative of melaninrich cells as cellular hypertrophy, cell process formation, and melanogenesis around the outside of nuclei. The enzymatic assay using L-DOPA and mushroom tyrosinase demonstrated that H2-Silica restrained UVA-mediated melanin formation owing to down-regulation of tyrosinase activity, which could be attributed to scavenging of free radicals and inhibition of L-DOPA-to-dopachrome oxidation by hydrogen released from H 2-Silica. Thus H2-Silica has a potential to prevent melanin production against UVA and serves as a skin-lightening ingredient for supplements or cosmetics. Copyright © 2013 American Scientific Publishers All rights reserved.

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