22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
The effect of cold plasma on human hepatocarcinoma cell and its 5-fluorauracil
resistant cell line
H. Yang, X.P. Lu and X. Yang
State Key Laboratory of Advanced Electromagnetic Engineering and Technology, Huazhong University of Science and
Technology, CN-430074 Wuhan, Hubei, P.R. China
Abstract: Atmospheric pressure cold plasma showed selective killing efficiency on cancer
cells in vitro and in vivo. However, the effects on chemotherapeutic drug resistant cell line
are rarely to be found. The effects of plasma on hepatocarcinoma cell line Bel7402 and its
5-fluorauracil resistant cell line Bel7402/5Fu were investigated. The results showed that
plasma induced superior toxicity to Bel7402 cells compared with Bel7402/5Fu cells.
Keywords: NTP (non-thermal plasma), liver cancer cells, MDR
1. Introduction
Atmospheric pressure cold plasma has been widely
applied in medicine and biology due to its own advantage
like generation of high concentrations of the chemically
active species while still maintaining room temperature
[1]. The reactive oxygen and nitrogen species (RONS)
produced by non-thermal plasmas exhibit strong oxidative
properties and trigger signalling pathways in biological
cells, the interaction of reactive species with biological
cells makes plasma possible for cancer therapy.
Atmospheric cold plasma induces cell growth arrest,
apoptosis and necrosis [2], meanwhile the dysfunction of
mitochondrial and DNA damage are also involved [3].
What’s more, it’s also been found that plasma has more
specific killing effect on cancer cells compared to normal
tissue cells.
Chemotherapy is one of the frontline strategies for the
treatment of cancer in clinic, but thousands of new cases
of cancer eventually exhibit multidrug resistance (MDR)
phenotype that either acquired from drug treatment or
intrinsic [4]. In this paper, the different effects and the
related explanation of plasma on hepatocarcinomal cell
line Bel7402 and its 5-fluorauracil resistant cell line
Bel7402/5Fu were conducted. And the reactive species
that were critical for the cytotoxicity were also evaluated.
2. Results
To elucidate the different effects of atmospheric plasma
on Bel7402 and Bel7402/5Fu cells, MTT assay as shown
Fig. 1 was used to detect the cytotoxicity of plasmatreated medium. These data suggested that Bel7402 cells
were much more sensitive to the plasma than
Bel7402/5Fu cells.
In order to explain the mechanism the effects of the
plasma on tumor cells, the ROS in the plasma-treated
medium contributing to the cell death was firstly
investigated. The results as shown Fig. 2 suggested that
hydrogen peroxide played the main role in the effects of
plasma-treated medium on cancer cells.
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Fig. 1. Effects of the plasma-exposed medium on the
viability of Bel7402 and Bel7402/5Fu cells.
Fig. 2. The effects of plasma-exposed medium on the
intracellular ROS level with the treatment of ROS
scavengers.
In order to understand what happened in Bel7402 and
Bel7402/5Fu cells, the intracellular ROS level as well as
the mitochondrial membrane potential after the incubation
with the plasma-exposed medium was studied (Fig. 3).
The relative fluorescence intensity increased in Bel7402
cells and the ROS-associated fluorescence decreased a
little bit.
The plasma treatment significantly decreased the ratio
of aggregates to monomers in Bel7402 cells and NAC
treatment promoted the recovery of the ratio, while
plasma treatment failed to affect on the Bel7402/5Fu
cells. These results indicated diverse effects of plasma
treatment on the intracellular ROS level and the
depolarization of the mitochondrial membrane potential
inBel7402 and Bel7402/5Fu cells (Fig. 4).
1
Bel7402 cells when compared to the control group.
Fig. 5. The transcriptional activityof AP-1, NF-кB and
p53 in plasma-treated Bel7402 and Bel7402/5Fu cells by
dual-luciferase expression assay.
Fig. 3. The intracellular ROS level in Bel7402 and
Bel7402/5Fu cells after incubation with plasma-treated
medium for 2 h.
Fig. 6. The intracellular catalase activity of Bel7402 (A)
and Bel7402/5Fu (B) cells.
Fig. 4. Mitochondrial membrane potential depolarization
in plasma-treated Bel7402 and Bel7402/5FU cells with
dye JC-1.
Since ROS can affect the expression, stability and
function of proteins, including transcription factors, the
disruption of the transactivation activity may inhibit the
target gene expression and thus modulate the ROS stress
and cell survival signalling [5]. To verify the effects of
the plasma-treated medium on the transcription factors,
the activities of AP-1, NF-кB and p53 were measured in
Bel7402 cells. As Fig. 5a showed, plasma-exposed
medium promoted the transcriptional activity of all three
transcription factors in a dose-dependent manner,
suggesting that they might played important roles in the
final apoptosis and necrosis intracellular signalling in
Bel7402 cell line. While as Fig. 5b showed, no obvious
trend has been measured in Bel7402/5Fu cells.
To investigate the intrinsic cause for the different
effects of plasma-treated medium on Bel7402 and
Bel7402/5Fu cells, the response of intracellular catalase
activity was explored. As shown in Fig. 6a, incubation
with medium pre-treated by plasma for 60 s induced
significant decrease in the intracellular catalase activity in
2
3. Discussion and conclusions
This study explored the inhibitory effects of
atmospheric pressure plasma on Bel7402 and its 5-Fu
resistant cell line. According to the MTT and apoptosis
assay results, it is obvious that the plasma-treated medium
caused significantly more cell death in Bel7402 cells than
Bel7402/5Fu cells. The plasma generated in He with O 2
and H2O is rich not only in O, O 2 * and O 3 , but also in
·OH and H 2 O 2 [6].
Hydrogen peroxide played an important role in the
cytotoxic effects on Bel7402 cells. The H 2 O 2 in the
medium led to the significant accumulation of
intracellular ROS, induced reactive oxygen stress
signaling outcome.
While in Bel7402/5Fu cells,
excessive increase in intracellular ROS levels as mediated
by plasma may induce the expression of endogenous
antioxidants.
Increase of antioxidants capacity in
Bel7402/5Fu endow the cells weapon to eliminate the
ROS stress brought by plasma treatment.
The cytotoxicity results showed that non-thermal
plasma might be implicated in the treatment of MDR
cancer cells, and attention should be paid to detailed
optimization and mechanism exploration.
The
accumulation of ROS generated by plasma either by
direct or separated approach in chemoresistant cells might
also affect the killing efficiency of the chemotherapeutic
drugs. The effects of plasma on sensitizing the MDR
cancer cells are still undergoing.
4. References
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Sons) (2013)
[2] C. Kim, et al. J. Biotechnol., 150, 4 (2010)
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A.J. Hak. PLoS ONE, 6, 11 (2011)
Q. Yin, et al. Adv. Drug Delivery Rev., 65, 13
(2013)
D. Trachootham, et al.
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D.X. Liu, et al. Appl. Phys. Lett., 98, 22 (2011)
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