Short Communication
ISSN: 2574 -1241

DOI: 10.26717/BJSTR.2019.19.003361

Antioxidant Effect of Hydrogen Nanobubble
Contributes to Suppression of Tumor Cell GrowthHiromi Kurokawa1, Hirofumi Matsui1, Hiromu Ito2, Atsushi Taninaka3, Hidemi Shigekawa3, Gjergj
Dodbiba4, Yuezhou Wei5 and Toyohisa Fujita5*
Faculty of Medicine, University of Tsukuba, Ibaraki, Japan1

Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan

2

Faculty of Pure and Applied Sciences, University of Tsukuba, Japan

3

Graduate School of Engineering, the University of Tokyo, Japan4College of Resources, Environment and Materials, Guangxi University, China

5*Corresponding author: Toyohisa Fujita, College of Resources, Environment and Materials, Guangxi University, China
ARTICLE INFO

Abstract

Received:

The antitumor effect of hydrogen nanobubble was evaluated in vitro and in vivo. Cancer
cell growth was inhibited in the hydrogen nanobubble-containing medium compared tothe non-containing medium (in vitro). Tumor growth was significantly suppressed in themiddle stage of transplantation (in vivo). We reported that hydrogen nanobubbles is a ROS
scavenger, therefore apoptosis due to ROS reduction may be induced in these results.

Published:July 15, 2019

July 22, 2019

Citation: Hiromi K, Hirofumi M, Hiromu
I, Atsushi T, Hidemi S, Gjergj D, Yuezhou
W, Toyohisa F. Antioxidant Effect ofHydrogen Nanobubble Contributes toSuppression of Tumor Cell Growth.
Biomed J Sci & Tech Res 19(5)-2019.
BJSTR. MS.ID.003361.

Keywords: Antioxidant; Hydrogen Nanobubble; Tumor Cell

Introduction
Hydrogen is known to have a strong antioxidant effect, derivedby its high reducing property [1]. However, hydrogen is difficult todissolve in water. Thus, an exclusive device is required to generate
the hydrogen water [2,3]. To clear this problem, we investigated thestable supply of hydrogen by nanobubbes. In our previously report,hydrogen nanobubbles eliminated the superoxide as compared toother gases in cell-free systems [4]. In this study, we examined theeffect of hydrogen nanobubbles on cancer growth in vitro and invivo.

Materials and Methods
Cell Culture
Rat cancerous gastric mucosa cell-line (RGK1) was establishedby exposing 1-Methyl-3-nitro-1-nitrosoguanidine to rat gastricmucosal cells. RGK1 was cultured in Dulbecco’s modified Eagles/

F12 medium without L-glutamine (Sigma-Aldrich Japan K.K.,Tokyo, Japan). Mouse Lewis lung carcinoma (LLC) cells wereobtained from Riken Cell Bank (Tukuba, Japan). LLC was cultured in
RPMI1640 medium (Wako Pure Chem. Ind., Ltd., Osaka, Japan). The
culture medium contained 10% heat-inactivated fetal bovine serum(Biowest, Kansas City, MO, U.S.A.) and 1% penicillin/streptomycin(Wako). Cells were cultured in a 37°C incubator in an atmosphereof 5% CO2 in air.

Preparation of H2-Medium and H2-Water

The 99.99999% hydrogen gas was bubbled into water for cell
culture (Millipore, Billerica, MA, U.S.A.) using a pore type porous
ceramic sparger, which has 500 nm of mean pore diameter for 30min. Using this water, H2-medium was prepared with Dulbecco’s
modified Eagles/F12 medium (Sigma-Aldrich) and 14.3 mM
NaHCO3 (Wako). H2-water was prepared using Gas & Water Double
Hydrogen Bottle® (WOO co., Ltd., Tokushima, Japan).

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Cell Viability Assay
Cell viability was examined using Cell Counting Kit-8 (CCK-8)(Dojindo, Tokyo, Japan) according to the manufacturer’s protocol.
RGK1 was cultured on 96-well plates at 2.5 × 103 cells/well andincubated overnight. The supernatant was aspirated, and themedium replaced to H2-medium. Cytotoxicity was determined byincubating cells at 37°C for 24h. Cells were incubated with 10%
CCK-8 reagent. Absorbance at 450 nm was measured by a DTX880
multi-mode microplate reader (Beckman Coulter, Brea, CA, USA).

Animal Protocol

Eight-week-old male C57BL/6 mice were used in vivo assay.
Mice were purchased from Japan SLC Inc. (Hamamatsu, Japan).

DOI: 10.26717/BJSTR.2019.19.003361

Animal protocol was approved by the University of TSUKUBAStanding Committee on Animals. Mice were injected in the flanksubcutaneously with LLC cells (2 × 106). After tumor implantation,
mice were obtained free access to water or H2-water. The tumor size
was measured at 13, 16, 19 and 22 days after tumor implantation.
The tumor volume was calculated as follows: Volume (mm3) =length x width2 x 0.5. On Day 22, mice were euthanized, and tumors
were calculated.

Results and Discussion

We investigated that the effect of H2-mediun for cancer cells.
Compare the culture medium (Control), cell viability of H2-medium
was significantly decreased (Figure 1A). Thus, H2-water can inhibit
cancer cell growth.

Figure 1: The effect of H2-medium or H2-watera)

Cell viability assay in RGK1.

b)Time dependent of tumor volume in tumor bearing mice.

The time dependent of tumor size in tumor bearing mice isshown in Figure 1B. The tumor size in the H2-water group was
smaller than that of the control group, and a significant difference
was observed at 19 days after tumor implantation. From this result,
H2-water can inhibit tumor growth. In this study, we indicated theanti-tumor effect of H2-medium and water in in vitro and in vivo. It
has been reported that tumor growth is suppressed by hydrogen
nanobubble [2,3]. Compared to these methods, our method is moreconvenient for producing nanobubbles using a pore type porous
ceramic sparger for hydrogen supply. We considered that we canprovide a more accessible method to obtain the antitumor effectby hydrogen nanobubbles. We have investigated the effects ofantioxidants such as monascus pigment and curcumin [5,6]. These
antioxidants inhibited the tumor growth. As a result of the reductionof acid ceramidase due to the reduction of ROS, it has been clarified
that the accumulation of ceramide is increased, thereby inducing
apoptosis [7-9]. Hydrogen nanobubbles are also considered to bedue to this mechanism because they have high scavenging abilityto superoxide.

Conclusion

The hydrogen nanobubbles induced inhibition of cell growth.
The mechanism of this effect suggested the induction of apoptosis
by ROS reduction.

Acknowledgement

This study was supported by Kakehashi projects in FY2018(TK18-05) and WOO co., Ltd.

References

1. Ohsawa I, Ishikawa M, Takahashi K, Watanabe M, Nishimaki K, etal. (2007) Hydrogen acts as a therapeutic antioxidant by selectively
reducing cytotoxic oxygen radicals. Nat Med 13(6): 688-694.
2. Saitoh Y, Okayasu H, Xiao L, Harata Y, Miwa N (2008) Neutral pHhydrogen-enriched electrolyzed water achieves tumor-preferential

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clonal growth inhibition over normal cells and tumor invasion inhibition
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5. Kurokawa H, Ito H, Matsui H (2017) Monascus purpureus induced
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DOI: 10.26717/BJSTR.2019.19.003361
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8. Seelan RS, Qian C, Yokomizo A, Bostwick DG, Smith DI, et al. (2000)
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ISSN: 2574-1241
DOI: 10.26717/BJSTR.2019.19.003361

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