22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Melanoma cells and singlet oxygen generated by electric discharge
A. Vodičková1, J. Schmiedberger2 and K. Kološtová3
1
Faculty of Biomedical Engineering, Czech Technical University in Prague, Prague, Czech Republic
2
Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
3 rd
3 Faculty of Medicine, Charles University, Prague, Czech Republic
Abstract: The aim of this study was to found out if singlet oxygen generated by discharge
can be used in medicine for melanoma treatment. In our study their influence on tumour
line B16 mouse melanoma is observed. The influence was evaluated by microscopy, MTT
assay, and xCELLigence System. The results of gene expression are already known, too.
Keywords: singlet oxygen, reactive oxygen species, electric discharge, DSOG, melanoma
cells
1. Introduction
In medicine the singlet oxygen is mainly used in the
photodynamic therapy. It is a combination of
photosensibilizator and the light with a wavelength
appropriate
to
absorption
maximum
of
the
photosensibilazator. Our experimental set-up produces
reactive oxygen species including singlet oxygen, by
radiofrequency/arc discharge without usage of any
chemicals or other devices. Since discharge-generated
singlet oxygen does not contain any dangerous chemicals
it should be perfectly suitable for the use in human
medicine. In our proof-of-concept experiments were used
melanoma cells B16 as the first biomedical samples.
Current shortcoming of the idea is low pressure in the
exposition chamber nevertheless results prove that it does
not affect used melanoma cells.
2. Experimental set-up
In our experimental set-up singlet oxygen is generated
by an electric discharge by an electron impact in DSOG-6
(Discharge Singlet Oxygen Generator), see Fig. 1. DSOG6 is a hybrid radiofrequency/arc (RF/DC) plasmatron [1].
The principle of producing singlet oxygen is fast mixing
Ar hybrid plasma jet with a neutral oxygen stream. The
detailed descriptions were published in [2, 3].
The biological samples were exposed in exposition
chamber where the low pressure (about 1300 Pa) and
room temperature are used.
3. Tumour line
For our experiments B16 melanoma tumour cell lines
were used. For the first sets of experiments the primary
melanoma cells were used and for the second experiment
the ascites and the circulating tumour cells were exposed.
Fig. 2. Picture of B16/PT (the photo was taken by Nikon
Eclipse TS 100).
The cells were incubated in 37 ˚C at 5% atmosphere of
CO 2 to 80% confluence. Then the singlet oxygen
exposures were carried out. The methodology of
experiments in illustrated in the Table 1.
Table 1. Methodology of experiments
Fig. 1. Scheme of DSOG-6 [1].
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Sample
Exposed to
Exposition time
1
reference
-
2
about 1300 Pa
10 min
3
O 2 + He:NO:Ar
10 min
4
O 2 (a1Δ g ) + O 2 :He:NO:Ar
2 min
5
O 2 (a1Δ g ) + O 2 :He:NO:Ar
4 min
6
O 2 (a1Δ g ) + O 2 :He:NO:Ar
6 min
7
O 2 (a1Δ g ) + O 2 :He:NO:Ar
8 min
8
O 2 (a1Δ g ) + O 2 :He:NO:Ar
9 min
9
ROS + O 2 :He:NO:Ar
4 min
1
Because in exposition chamber there is low pressure, in
each set of experiments we observed the influence of low
pressure on the tumour cells lines.
Evaluation methods were light microscopy, MTT assay
and xCELLigence system. After all experiments the gene
expression by real-time qRT-PCR was performed.
4. Results of experiments
At first the cell cultures were observed in a light
microscope. Living cells are adhesive, therefore they are
on the bottom of the cultivation flask or Petri dishes. But
non-living or dead cells are floating in the cultivation
medium. From microscopy we found out that ascites and
circulating tumour cells are more sensitive to exposure to
singlet oxygen than melanoma cells without expositions.
Fig. 3. The cells after exposition to singlet oxygen. The
dark blue cells are apoptotic.
MTT assay is colorimetric assay for testing to cytostatic
cisplatine in our case. Cisplatine is commonly used for
skin cancer treatment. We compared singlet oxygen
therapy and combination of singlet oxygen therapy and
chemotherapy. xCELLigence system is a method for realtime monitoring of cell viability. We also tested
sensitivity to cisplatine by this assay, but in real time.
Fig. 4. Chart evaluated by SW RTCA. Red line illustrates
B16/Pt exposed to singlet oxygen for 8 minutes. Green
line illustrates cells exposed 8 minutes to singlet oxygen
and added cisplatine. Blue line illustrates exposition to
singlet oxygen for 2 minutes and violet line is the same
sample with cisplatine added.
From Fig. 4 it is clear that cells exposed to singlet
oxygen are dying, but cells exposed to singlet oxygen and
in combination with cisplatine are living. xCELLigence
system was used for verifying of MTT assay. From these
assays we found out that cells exposed to singlet oxygen
2
are more resistive to cisplatine. So these therapies can´t be
combined.
Gene expression was the last step of our preliminary
singlet oxygen study. For this evaluation we used realtime qRT PCR technique (real-time quantitative Reverse
Transcription Polymerase Chain Reaction) and mouse
TaqMan® probes. We chose these gene primers: ACTB
(actin) is gene coding of actin. MLANA (melan A) is
gene of melanoma cells. CASP3 (caspase -3) is gene
which has influence on cell apoptosis. BCL2 (B-cell
LCC/lymphoma2) is apoptosis regulator and BIRC5
(survivin) is inhibitor of apoptosis. HIF1 (hypoxia
inducible factor), VEGF (vascular endothelial grown
factor) and KDR (kinase insert domain receptor) are
genes for hypoxia. COX2 (cytochrome C oxidase
assembly factor) and NFKB1 (nuclear factor of kappa
light polypeptide gene enhancer in B-cells) are genes for
inflammation.
Results of gene expression are under investigations.
5. Conclusion
In preliminary experiments melanoma cells (primary,
ascites and circulating melanoma cells) were exposed to
singlet oxygen and a few samples to reactive singlet
oxygen species. We found out that cisplatine therapy and
singlet oxygen therapy cannot be combined, because of
higher resistance of cells exposed to singlet oxygen to
cisplatine. The result of gene expression is still under
investigations. However new questions has rised up. In
the planned future experiments we would like to make
experiment with melanoma cells exposed to singlet
oxygen and to reactive oxygen species in same time long
interval and compare influences of these radicals.
4. References
[1] Schmiedberger, J., Rohlena, K., Gregor, J., Křenek, P.,
Jirásek, V., Čenský, M., and Kodymová, J., “Hybrid
RF/DC plasma torch for generation of singlet oxygen
in discharge oxygen-iodine laser“, Proceedings SPIE,
Vol. 7751, 775110G-1-9, (2010).
[2] A. Vodičková and J. Schmiedberger, “Proposal of
Biomedical Applications of Singlet Oxygen Generated by
Electric Discharge”, Conference on Instruments and
Methods for Biology and Medicine 2011, Faculty of
Biomedical Engineering CTU in Prague, June 2,
Proceedings 77-80, (2011).
[3] Schmiedberger J., Gregor J., Jirásek V., Čenský M.,
Rohlena K. and Vodičková A., “Recent characterization
of hybrid radiofrequency/arc discharge for generation of
singlet oxygen”, 21th International Symposium on Plasma
Chemistry, 4th-9th August 2013, Cairns Convention
Centre, Australia, Proceedings (2013).
5. Other Specifications
This work has been supported by the Czech Science
Foundation under the grant project P102/12/0723.
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