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Growth Suppression of HL60 and L6 Cells by Atomic Hydrogen

  • Kensuke Nakanishi
  • Takeki Hamasaki
  • Takuro Nakamura
  • Masumi Abe
  • Kiichiro Teruya
  • Yoshinori Katakura
  • Shinkatsu Morisawa
  • Sanetaka ShirahataEmail author
Conference paper
Part of the Animal Cell Technology: Basic & Applied Aspects book series (ANICELLTECH, volume 16)

Abstract

We previously reported electrolyzed reduced water (ERW) which produced near the cathode by electrolysis has a reductive activity. We also revealed that ERW contains both molecular hydrogen and platinum nanoparticles (Pt nps) derived from platinum-coated titanium electrodes. Pt nps have catalysis activity converting molecular hydrogen to atomic hydrogen. Thus, we regard a solution containing both dissolved hydrogen and Pt nps as a model water of ERW (H2/Pt nps solution), and then we tried to examine the physiological effects of H2/Pt nps solution. To our knowledge, there is no report on physiological effects of H2/Pt nps solution. Here, we report the effects of H2/Pt nps solution on the growth of promyelocytic cell line, HL60 cells and myogenic cell lines, L6 cells. H2/Pt nps solution suppressed cell growth in the presence of both hydrogen and Pt nps in a dose dependent manner. The result of sub-G1 analysis suggests that H2/Pt nps solution induces apoptosis in both HL60 and L6 cells. The labeled Pt nps in HL60 cells were detected in liposome, suggesting Pt nps is incorporated by endocytosis. These results suggest that atomic hydrogen catalyzed by Pt nps induced significant cell growth suppression and atomic hydrogen may be one of new signal of cell function.

Keywords

Platinum Nanoparticles Apoptosis Atomic hydrogen Antioxidant ROS 

1 Introduction

We previously reported ERW which is produced near the cathode by electrolysis has a reductive activity and tumor-suppressive activity [1, 2, 3]. We also revealed that ERW contains both molecular hydrogen and Pt nanoparticles (nps) derived from platinum-coated titanium electrodes. Synthesized Pt nps have catalytic activity converting molecular hydrogen to atomic hydrogen. Consequently, there is a possibility that the atomic hydrogen is produced in ERW. Thus, we synthesized Pt nps to construct a model water of ERW containing both molecular hydrogen and Pt nps, and then we examined the effect of the model water on the cell viability and Sub-G1 analysis in this study.

2 Experimental Procedure

2.1 Cell Culture and Measurement of Cell Proliferation

Premyeloblastic cell lines (HL60; JCRB0085), skeletal muscle myoblast cell lines (L6; JCRB9081) were obtained from the Japanese Collection of Research Bioresources (JCRB, Osaka, Japan). HL60 Cells were cultured in RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) (Invitrogen Japan K. K., Tokyo Japan) at 37°C in a 95% air/ 5% CO2 atmosphere (10% FBS/MEM).L6 cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS at 37°C in a 95% air/ 5% CO2 atmosphere. Briefly, cells were seeded (floating cells, 1.0 × 105 cells/cm2 and adherent cells, 2.0 × 104 cells/cm2) in 6-well plates. The cultures were incubated with different concentrations of Pt nps for 2 h in serum-free medium, washed two times with PBS, exchanged with fresh serum containing medium and incubated with a gas incubator at 37°C in a 75% H2/ 20% O2/ 5% CO2 atmosphere for 24 h. Dissolved helium solution was used as control. The number of cells was counted by a cell counter.

2.2 Cell Cycle Analysis by Flowcytometer

Cells were harvested 2 h after treatment with or without H2/Pt nps solution. After washing with PBS, 1×106 cell pellets were suspended in 0.5 ml of hypotonic fluorochrome solution, containing 50 μg/ml propidium iodide (PI; Sigma, MO). Cells were stained overnight in the dark at 4°C and examined by a flowcytometer (EPICS XL ADC System, Beckman Coulter, Inc. Ca, USA).

3 Results and Discussion

Cells were incubated with dissolved H2 solution after treatment with Pt nps at 2 h in FBS-free medium. The cell viability was decreased on both HL60 and L6 cells when supplemented with both Pt nps and H2 (Fig. 1). The result of trypan blue dye assay shows that this suppression of cell viability was caused by cell death (data not shown). Thus, we next examined the cell death by Sub G-1 analysis using flowcytometer.
Fig. 1

Dissolved hydrogen solution supplement with Pt nps suppresses the growth of HL60 cells and L6 cells. Bars represent means ±SD. * P<0.01 in comparison with control

Increasing of Sub-G1 phase is one of the indications of apoptosis. Analysis of cell cycles was performed 24 h after Pt nps supplement. In the condition of dissolved hydrogen solution supplemented with 3.0–5.0 ppm Pt nps in HL60 cells and L6 cells, 20–37% of Sub-G1 phase are increased compared to control medium. The result suggests that atomic hydrogen induced apoptosis of both L6 and HL60 cells (Fig. 2 ).
Fig. 2

Cell cycle analysis of HL60 and L60 cells cultured in medium supplemented with H2 and Pt nps

The H-transfer reaction in vitro by antioxidants has been well investigated so far. There are many reports about the activities of antioxidants. However, there are few reports about effects in a living organism of atomic hydrogen itself. We expect that physiological effects of atomic hydrogen itself will be figured out in the future.

References

  1. 1.
    Shirahata, S., Kabayama, S., Nakano, M., Miura, T., Kusumoto, K., Gotoh, M., Hayashi, H., Otsubo, K., Morisawa, S., Katakura, Y. (1997). Electrolyzed-reduced water scavenges active oxygen species and protects DNA from oxidative damage. Biochem. Biophys. Res. Commun. 234: 269–274.CrossRefPubMedGoogle Scholar
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    Shirahata, S. Reduced water for prevention of diseases. In: Shirahata, S. Teruya, K. Katakura, Y., (Eds.) (2002) Animal Cell Technology: Basic & Applied Aspects (Volume 12, pp. 25–30). Netherlands: Dordrecht. Kluwer Academic Publishers.Google Scholar
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    Huang, K. C., Yang, C. C., Lee, K. T., Chien, C. T. (2003) Reduced hemodialysis-induced oxidative stress in end-stage renal disease patients by electrolyzed reduced water. Kidney Int. 64: 704–714.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Kensuke Nakanishi
    • 1
  • Takeki Hamasaki
    • 1
  • Takuro Nakamura
    • 1
  • Masumi Abe
    • 1
  • Kiichiro Teruya
    • 2
    • 3
    • 4
  • Yoshinori Katakura
    • 5
    • 6
    • 7
  • Shinkatsu Morisawa
    • 8
  • Sanetaka Shirahata
    • 5
    Email author
  1. 1.Department of Genetic Resources TechnologyKyushu UniversityFukuokaJapan
  2. 2.Graduate School of Systems Life Sciences, Kyushu UniversityHigashi-kuJapan
  3. 3.Department of Genetic Resources Technology, Faculty of AgricultureKyushu UniversityFukuokaJapan
  4. 4.Graduate School of Bioresource and Bioenvironmental SciencesKyushu UniversityFukuokaJapan
  5. 5.Faculty of AgricultureKyushu UniversityHigashi-kuJapan
  6. 6.Graduate School of System Life ScienceKyushu UniversityFukuokaJapan
  7. 7.Department of Genetic Resources TechnologyKyushu UniversityFukuokaJapan
  8. 8.Nihon Trim co. LTDOsakaJapan

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