22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Non-thermal atmospheric pressure plasma induces selective apoptosis in
p53-deficient cancer cells and efficiently kills CD133+ cancer stem cells
depending on the source of gas supply
K. Song1, G. Li1, Y. Ma1, E.H. Choi2 and H.J. Lee3
1
Department of Biochemistry, Yonsei University, Seoul, Korea
Department of Electrophysics, Kwangwoon University, Seoul, Korea
3
Department of Electrical Engineering, Pusan National University, Pusan, Korea
2
Abstract: Non-thermal atmospheric pressure plasma (NTAPP) is defined as a partially
ionized gas-containing electrically charged particles. Because of its low temperature,
NTAPP has promising biomedical applications, such as wound healing and sterilization.
Many recent studies have shown that NTAPP strongly induces apoptosis in cancer cells by
generating reactive oxygen species (ROS). In our study, we examined the apoptotic effect
of NTAPP generated from helium, nitrogen and air. We first used helium-based NTAPPgenerating apparatus to treat p53 wild type (LoVo, MES-SA, HepG2, RKO) and
p53-deficient (DLD-1, H1299, HT29, HCT115) cancer cell lines, primary cells and cancer
stem cells (CSCs). We found that repetitive exposures of NTAPP preferentially induce
apoptosis in cancer cells, especially in p53-deficient cells, but not in primary and cancer
stem cells. In order to use NTAPP to induce the apoptosis of CSCs, we developed a new
NTAPP-generating device with air and nitrogen supply, and examined the apoptotic effect
of NTAPP generated in this device in Huh7 (p53-mutated), Hep3B (p53-deleted), and
HepG2 (p53-proficient) cells that express liver CSC positive markers CD133, ALDH1, and
EpCAM. When we used the same NTAPP exposure condition as the helium-based device,
we found that both nitrogen- and air-based NTAPP treatment efficiently decreased the cell
viability of HepG2, Hep3B, and Huh7.
Key words: non-thermal atmospheric plasma, apoptosis, cancer, p53, cancer stem cells
1. Introduction
Non-thermal atmospheric plasma (NTAPP) provides
promising applications in biomedical research. Evidence
from many research groups suggests that NTAPP can be
safely and controllably applied to animals and humans.
NTAPP has been used in sterilization of different surfaces
of living tissues, water, and air. In addition, it has
potential applications in would healing [1]. Recently,
several groups have reported that NTAPP can induce
apoptosis selectively in cancer cells [2] [3] 4], suggesting
its potential in cancer therapy. ROS and RNS have been
reported to play pivotal roles for the biomedical effect of
NTAPP. The composition and concentration of NTAPP
vary depending on the device and gas used to generate it.
Cancer is a group of diseases involving uncontrollable
cell proliferation. Most cancer cells have lost abilities to
control cell cycle or repair system. More than 50% of
human cancer cells have p53 gene dysfunction, either
mutation or deficiency.
In addition to focusing on critical gene mutations found
in cancer cells, more scientists now are turning their
attention to a new group of cells exist in tumors, which
are called cancer stem cells. Cancer stem cell (CSC) or
tumor-initiating cell (TSC) is defined as a kind of cancer
cell that possesses the capacity of self-renewal to maintain
the tumor size [5]. CSCs have three important properties:
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self-renewal and promoted tumor heterogeneity, drug
resistance, and metastasis, making them highly resistant
to existing cancer therapies. Although specific biomarkers
in CSCs are not clear, some markers have been suggested
including CD133, CD44, ALDH1, and EpCAM.
Increasing number of cancer cell lines with self-renewal
and cancer-initiating capability has been reported to carry
CD133.
2. Results
In order to verify the effect of NTAPP on cancer cells,
we designed a helium-based dielectric barrier discharge
(DBD) device for NTAPP exposure to cells. The scheme
of DBD device is illustrated in Figure 1A. We then treat
several p53 wild type (LoVo, MES-SA, HepG2, RKO)
and p53-deficient (DLD-1, H1299, HT29, HCT115)
cancer cell lines with repetitive exposures of NTAPP. As
shown in Figure 1B, the anti-proliferative effect of
NTAPP was more efficient in p53-deficient cells than p53
wild type cells. This result strongly suggests that NTAPP
has a selective anti-proliferative effect on p53-deficient
cells. To further verify our conclusion, we transfected
HT29 cells, a p53-deficient cell lines, with p53 expressing
vector. After treated with repetitive NTAPP, the cell
viability of HT29 with empty vector decreased
dramatically, while the cell viability was recovered after
1
p53 gene transfection (Figure 1C).
To analyze the effect of NTAPP on CSCs, we
repetitively exposed HepG2, Hep3B and Huh7, the liver
cancer cells that express high level of CD133, to NTAPP
with the same helium-based device. The percentage of
viable cells in Huh7 was decreased when compared with
non-treated control, but the relative percentage of viable
cells were increased after 72 h incubation (Figure 1D),
suggesting that there was no severe cell death effect. To
verify whether NTAPP-treated Huh7 cells undergo
apoptosis, we examined the levels of DNA double-strand
break responding protein, γ-H2AX and PARP, a
downstream marker of apoptosis in NTAPP-exposed
Huh7 cells. We observed that NTAPP exposures could not
induce apoptosis in Huh7, while the same exposures of
NTAPP induced effective cell death in cancer cells
(Figures 1E).
Fig. 1. Helium-based NTAPP selectively induce apoptosis of p53-deficient cells but not liver cancer stem cells.
A) Schematic description of the NTAPP-generating device for applying NTAPP to living cells. B) The relative
percentage of viable cells after the same NTAPP exposures in p53-proficient cells (RKO, MES-SA, HepG2, G361,
LoVo) and p53-deficient cells (DLD-1, H1299, HT29, HCT115) by MTT assays. C) The relative percentage of
viable cells of HT29 and p53-transfected HT29 cells by MTT assays after NTAPP treatment. The expression of
p53 in HT29 was verified by western blot shown on the right side. D) Huh7 CD133-positive cells were treated
with helium-based NTAPP and cell viability was measured by MTT assays. E) Western blot analyses of γ-H2AX
and PARP in Huh7 cells following NTAPP treatment.
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22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Fig. 2. Nitrogen-based NTAPP efficiently decreases the viability of liver cancer stem cells.
A) Schematic description of the NTAPP-generating system to treat live cells in vitro. B) Expression of cancer stem
cell markers, CD133, EpCAM, and ALDH1 was analysed by reverse transcriptional PCR in Huh7, HepG2, and
Hep3B cells. C) Huh7, HepG2, and Hep3B cells were exposed to nitrogen-based NTAPP for 60 s per each hour for
10 times and further incubated. Cell viability was measured by MTT assays after 9, 24, 48 and 72 from the first
NTAPP treatment.
Since helium-based NTAPP device did not induce
apoptosis in CSC, we designed another DBD type NTAPP
device that use both air and nitrogen as a gas supply
(Figure 2A). Before we examined the effect of NTAPP to
CSCs, we first analyzed the expression of wellestablished liver CSC markers, CD133, EpCAM, and
ALDH1 in HepG2, Hep3B, Huh7 cells. The expression of
EpCAM, ALDH1, CD133, and the control GADPH in
these CSCs were tested in Figure 2B by reverse
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transcriptional PCR. Then, HepG2, Hep3B, Huh7 cells
were treated with both nitrogen- and air-based NTAPP for
60 s per every hour for 10 times and cultured for 24, 48,
and 72 h. Cell viability of all three CSCs decreased
dramatically when exposed to NTAPP for 60 s per every
hour for 10 times, when either nitrogen or air was used as
a gas source (Figure 2C and data not shown). These
results demonstrated that NTAPP generated by the new
device efficiently induce the death of CSCs.
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22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
3. Discussion
We observed that NTAPP exposures induce cell death
much more efficiently in p53-mutated cancer cells than in
p53 wild-type cells. We confirmed this results by
examining apoptotic cell death by NTAPP in several
different types of cancer cells containing either wild-type
p53 or mutated p53. We further confirmed this result by
demonstrating that the viability of p53-deficient HT29
cells that were highly sensitive to NTAPP became
recovered when p53 was transfected. Several studies
reported that tumor suppressor p53 plays pivotal
important role in protecting the genome from various
genotoxic and cellular stress especially in oxidative stress.
When cells are under oxidative stress, p53 becomes
activated and stabilized and regulates the expression of a
number of targeting genes to activate antioxidant, arrest
cell cycle and repair of the damaged DNA lesion. More
than 50% of human cancer cells have p53 gene mutations
that lead to defects in the function of p53. So our results
that NTAPP preferentially induces apoptosis in p53mutated cancer cells support the potential of NTAPP as a
cancer therapy with a significant advantage.
The CSCs are known to be highly resistant to chemoand radiation therapies. Thus, we questioned whether
NTAPP efficiently induces cell death to be used as an
effective therapy against CSCs. In this study, we used
HepG2, Hep3B, and Huh7 liver cancer stem cells,
HepG2, Hep3B, and Huh7 that express different levels of
the cancer stem cells markers and different p53 status:
HepG2 is p53-wild type cells with a lowest CD133
expression, Hep3B is p53-deficient with highest CD133,
Huh7 is p53-mutant with the moderate expression of
CD133 [6]. Air- or nitrogen- based NTAPP efficiently
induced dramatic decrease in viable cells, demonstrating
that NTAPP is highly efficient to kill all three CSCs
regardless of the CD133 expression level and the p53
status. These observations suggest that p53 is not a crucial
factor for inducing cell death in cancer stem cells, while
p53-deficient cancer cells are highly sensitive to NTAPP
treatment.
Previous studies have shown that different gases used
for NTAPP may have different effects on inducing
apoptosis. In our study, we used a helium-based device
and a nitrogen or air-based device to treat CSCs. In short,
we demonstrate that air or nitrogen-based NTAPP is more
effective to decrease the cell viability of liver CSCs
compared with helium-based NTAPP.
4. Reference
1. Isbary G, Clinical Plasma Medicine. 1 (2) (2013).
2. Huang J, Applied Physics Letters. 99 (2011).
3. Yan X, Plasma Processes and Polymers. 9 (1) (2012).
4. Ishaq M, Molecular biology of the cell. 25 (9) (2014).
5. Clarke MF, Cancer research. 66 (19) (2006)
6. Zhu Z, International journal of cancer Journal
international du cancer. 126 (9) (2010).
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