st
21 International Symposium on Plasma Chemistry (ISPC 21)
Sunday 4 August – Friday 9 August 2013
Cairns Convention Centre, Queensland, Australia
Gene ontology analysis for HeLa cells exposed to
a plasma- or H2O2- treated culture medium
Takehiko Sato1, Mayo Yokoyama1 and Kohei Johkura2
1
Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan
Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621,
Japan
2
Abstract: To find particular biological responses by exposure to plasma-treated culture, a
feasibility study for investigating HeLa cell response to the plasma- or H2O2-treated culture
medium was carried out with a gene ontology analysis. We found that the particular biological
responses of HeLa cells are expressed by exposure to the plasma-treated culture medium by
the gene ontology analysis.
Keywords: Gene ontology analysis, Plasma-treated culture medium, biological response
1. Introduction
Recently, plasma treatments for inactivation of microorganisms such as pathogenic bacteria and virus are remarked to prevent infections in hospitals, public space
and home because plasma can generate chemically reactive species, light, heat, electric fields and shock waves at
a low cost, safety and a dry process [1-12]. Plasma treatment is pushed forward to apply to medical treatments
such as skin diseases and blood coagulation and a microwave argon plasma for skin diseases has been tested clinically[1,2]. Plasma stimuli show some good effects on the
healing which are caused by reduction of suppuration,
inflammation by the sterilization of the wound part, and
cell proliferation by cell activation by NO gas. For example, Max-Planck-Institute group has clarified that various
bacteria reduced enough in 38 clinical cases for the case
of chronic wounds which are difficult to heal by conventional treatments [3]. They have already tested more than
2000 treatments to develop practical products. The group
of Shekter and others reported that the healing enhancement was obtained by the plasma-generated NO of
300ppm or 500ppm exposure to the wounds. They have
already tested for 318 patients and the reduction of
wounds area decreased 1.7%/day in the group of NO
treatment though it was 0.7%/day in the control group.
Argon Plasma Coagulation (APC) is a commercial
product which can stop bleeding by plasma heating [13].
The APC which had been built in an endoscope was developed in 1994 [14] and it is widely used in the world for
blood stanching of digestive tracts, coagulation operation
for esophagus tumor and treatments of nasal allergy. This
equipment is capable of selecting automatically
non-coagulated blood and cauterize locally. After coagulation, the plasma discharge to the other non-coagulated
blood immediately. Therefore, it can treat with low heat
damage. Now, new low-temperature plasma coagulation
devices using chemical reactions are developing [15].
It is reported that the apoptotic instruction to a cancer
cell by plasma irradiation [16-18], as for the plasma med-
icine, the application to a next-generation cancer therapy
is expected. However, the mechanism of apoptosis induction is almost unknown due to generation of a variety of
physical stimulation by plasma. Particularly, the distinction between the effects of radicals and ions which have
short life spans, and the effects of the stable chemical
species are still difficult though such radicals are immediately deactivated and change to more stable chemical species such as H2O2, HNO3, HNO2 in a short time underwater.
Authors have clarified that an effect of these stable
chemical species on HeLa cell viability [19]. Inactivation
of HeLa cell viability was not shown in the cases of
HNO3 and HNO2 in the range of the quantity that plasma
generated. On the other hand, inactivation effect on HeLa
cell viability showed a similar trend between plasma-treated and H2O2-added culture media. Here, the cell
survival rate to H2O2-added (304 µM) was 89%, 76%,
13% and 0% in 10 min, 30 min, 60 min and 120 min respectively. Correspondingly, in the case of plasma-210
s-treated, the cell survival rates were 93%, 83%, 15% and
0%. In addition, by morphological observation, authors
showed that the cells started to shrink after 4-5 hours,
then blebs appeared after 5-6 hours and finally cells were
inactivated in both cases of exposure to the plasma-treated
and H2O2 -added culture media. The reactive oxygen
concentration in the cells and the cell survival rate in the
case of catalase-added also show the same trend between
the plasma-treated and H2O2-added culture media. These
results evidenced that the key inactivation factor of HeLa
cells is H2O2 among stable chemical species generated by
plasma.
In this study, we focus on revealing what kind of cytotoxicity is generated in the plasma-treated culture medium
and how cells respond from a viewpoint of gene expressions using a comprehensive gene analysis method.
st
21 International Symposium on Plasma Chemistry (ISPC 21)
Sunday 4 August – Friday 9 August 2013
Cairns Convention Centre, Queensland, Australia
Fig. 2 Number of genes which showed different expression
levels between the two conditions in each group.
Multicellular
organismal
process 10%
Fig. 1 Experimental procedure
2. Experimental methods
Plasma flow was generated in the 1.5-mm gap between
a pin electrode and the surface of the culture medium as
shown in Fig. 1. The electrode was made of platinum and
was 0.3 mm in diameter. Culture medium of 1 ml in a
1.5-ml microtube was set in a ground electrode. A voltage
of +7.5 kV0p with a frequency of 5 kHz and a duty ratio of
4% was applied to the pin electrode for 210 s. The chemical species such as NO and excited N2 were observed by
the spectrum analysis and those species should be dissolved in the culture medium and considered to change to
HNO2 and HNO3. The H2O2 concentration in the plasma-treated culture medium was measured by calibration
with the H2O2 absorbance curve against the known concentration in the culture medium. The H2O2 reaction reagent can discolor the solution depending on H2O2 concentration. The H2O2 concentration was 304 µM when it was
measured in the culture medium treated by the plasma
flow for 210 s.
The HeLa cells (obtained from Cell Resource Center
for Biomedical Research, Institute of Development, Aging
and Cancer, Tohoku University) were cultured with a regular medium which consists of Minimum Essential Medium (MEM) (SIGMA), 10% Fetal Bovine Serum (FBS)
(GIBCO) and Penicillin-Streptomycin (Penicillin 100
unit/ml; streptomycin 100 μg/ml) (Nacalai Tesque). The
cells were incubated in a flask of 25 cm2 at 37ºC and
5%CO2 for 23 hr. Comprehensive gene expression analysis (Whole Human Genome Microarray Kit, Agilent
Technologies) was carried out by the Dragon Genomics
Center, Takara Bio Inc. The gene ontology analysis was
performed by a GeneSpring software (Agilent Technologies). The DNA micro array used in this study can ana-
Developmental
process
10%
Cellular
Process
32%
Locomotion
7%
Response to
stimulus
13%
Biological
regulation
25%
Fig. 3 Pie chart for the biological process in the case of (1)
Plasma 210s/Control, expression ratio < 0.5．
lyze 32,000 kinds of genes at the same time.
3. Results and discussion
In microarray analysis of HeLa cell response, the treatment conditions for culture media were as follows: control (C), plasma exposure for 210 s (P210s), addition of
H2O2 with 304 µM (H304µM), plasma exposure for 40 s
(P40s) and addition of H2O2 with 51 µM (H51µM). The
gene expression levels were compared in two conditions
of eight sets: (1) P210s/C, (2) H304µM/C, (3) P40s/C, (4)
H51µM/C, (5) P210s/H304µM, (6) P210s/P40s, (7)
P40s/H51µM and (8) H304µM/H51µM. Around 32,000
genes analyzed, the number of genes with an expression
ratio ‘equal to or greater than 2’ or ‘equal to or less than
0.5’ were counted in each comparison as shown in Fig. 2.
The number of the former case was prominent in 3 and 4,
and correspondingly that of the latter case was also notable in 6 and 8. These results implied that the effect of
plasma-treated culture medium on the gene expressions of
HeLa cells was similar to that of H2O2.
Figure 3 shows the Pie chart of large categories for ‘expression ratio < 0.5’ in Plasma 210s/Control (case 1 in Fig.
2). As indicated by Cellular process (32%) and Biological
regulation (25%), gene expressions involved in
st
21 International Symposium on Plasma Chemistry (ISPC 21)
Sunday 4 August – Friday 9 August 2013
Cairns Convention Centre, Queensland, Australia
Fig. 4 The percentage of genes belonging to each term of Cellular component. Plasma (210s)- or H2O2 (304
µM)-treatment was compared with control, and up-regulated genes with an expression ratio equal to or greater than
2 were evaluated.
metabolism, proliferation, and intercellular signaling was
shown to be decreased by plasma treatment. . In addition,
the difference between plasma (210s) and H2O2 (304µM)
appeared in biological adhesion, cell adhesion, cell migration, cell motility, localization of cell, and locomotion,
which shows that gene expressions involved in cellular
adhesion and locomotion were altered.
We also found that there were differences between them
in particular terms for Cellular component. As shown in
Fig. 4, the differences were evident in Cellular component,
which describes locations of gene products. The percentage of up-regulated genes in the plasma-treatment was
much greater than that in the H2O2-treatment for the
components of membrane part, intrinsic to plasma membrane and plasma membrane part. In contrast, the percentage was larger in the H2O2-treated case for extracellular region, extracellular space and extracellular region
part. We also revealed the different responses in Biological process and Molecular function between them. These
results imply that the particular biological responses of
HeLa cells are expressed by exposure to the plasma-treated culture medium.
4. Conclusions
This study investigated the effects of chemical species
generated by plasma exposure to culture medium and
H2O2 added culture medium on cell responses from the
point of gene expression. By the comprehensive gene
analysis, it was revealed that there are differences between plasma-treated and H2O2-added on the components
of membrane part, intrinsic to plasma membrane and
plasma membrane part in the terms for Cellular component though the general effect on gene expression of
HeLa cells was similar. Plasma-treated culture medium
showed the inactivation of the gene expression for the
metabolism, proliferation, and signal between cells.
5. Acknowlegements
This study was partly supported by the Japan Society
for the Promotion of Science, Grant-in-Aid for Scientific
Research, and by Collaborative Research Project of the
Institute of Fluid Science, Tohoku University. We would
like to thank D. Yoshino and T. Nakajima, Tohoku University for technical support and discussion.
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