Int J Clin Exp Pathol 2016;9(2):1061-1067
www.ijcep.com /ISSN:1936-2625/IJCEP0019576

Original Article
Modulated expression levels of tyrosine kinases inspontaneously developed melanoma by single
irradiation of non-thermal atmosphericpressure plasmas
Yasuhiro Omata1*, Machiko Iida1*, Ichiro Yajima1, Nobutaka Ohgami1, Masao Maeda1, Hiromasa Ninomiya1,
Reina Oshino1, Toyonori Tsuzuki2, Masaru Hori3, Masashi Kato1
Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, 65
Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan; 2Department of Pathology, Nagoya Daini Red Cross
Hospital, Showa-ku, Nagoya, Aichi 466-8650, Japan; 3Department of Electrical Engineering and Computer Science, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan. *Equal
contributors.
1

Received November 11, 2015; Accepted January 12, 2016; Epub February 1, 2016; Published February 15, 2016
Abstract: Development of therapy for melanomas without BRAFV600E mutation, which account for about half of allmelanomas, is an urgent issue because effective therapy is currently being developed for patients who have melanomas with BRAFV600E mutation. RET-transgenic mice (RET-mice) carrying the RFP-RET oncogene under the controlof metallothionein-I promoter spontaneously developed skin melanomas without BrafV600E mutation from benign
melanocytic tumors. We previously showed decreased expression levels of cell cycle regulators and matrix metalloproteinases in melanoma from RET-transgenic mice by single irradiation of non-equilibrium atmospheric pressureplasmas (NEAPPs). In this study, we focused on RFP-RET, c-Ret, Epidermal growth factor receptor (Egfr), Vascularendothelial growth factor receptor 2 (Vegfr2) and c-Src kinases, which are correlated with melanoma. We first confirmed significantly increased transcript expression levels of the 5 kinases in melanomas compared to those inbenign tumors in RET-mice. We then found that transcript expression levels of c-Ret and Egfr, but not those of RFPRET, Vegfr2 and c-Src, were significantly decreased by single irradiation of NEAPP. Since EGFR-mediated promotionof melanoma has been reported, we further focused on the mechanism of NEAPP-mediated decrease in the levelof Egfr. Transcript expression level of Y box protein 1 (Ybx1), but not those of p53, Early growth factor 1 (Egr1), GCrich sequence DNA binding factor 2 (Gcf2) and Kluppel-like factor 10 (Klf10), was significantly decreased by single
irradiation of NEAPP. These results suggest that NEAPP decreased Egfr expression level through decrease of Ybx1
expression. Our results indicate that NEAPP irradiation to melanoma without BRAFV600E mutation is a possible novel
therapy.
Keywords: Non-thermal atmospheric pressure plasmas (NEAPPs), melanoma, tyrosine kinase, Epidermal growth
factor receptor (Egfr), Y box protein 1 (Ybx1)

Introduction
The worldwide incidence of melanoma, themost aggressive cutaneous cancer, has recently become higher than that of any other cancer.
Recent molecular-targeted therapy for melanomas with BRAF V600E mutation is developing inhumans despite recurrence of the melanomas[1]. In fact, the prognosis of patients who havemelanoma with BRAF V600E mutation is better
than that of patients who have melanoma with-out BRAF V600E mutation [2]. Therefore, the development of a novel therapy for melanomas
without BRAF V600E mutation, which account for
about half of all melanomas, is an urgent issue.
Previous studies showed that protein tyrosine
kinases (PTKs) play a crucial role in various cancers including melanoma. c-Ret/RET protein isa receptor-type PTK and is activated by dimer
formation through its ligands such as a glial cellline-derived neurotrophic factor (GDNF) [3, 4].

Effect of plasma irradiation on expression of tyrosine kinase genes

Figure 1. Macroscopic and microscopic appearances of tumors in RET-mice. (A-D) Representative macroscopic (A,C) and microscopic (B, D) appearances of a cutaneous benign melanocytic tumor (A, B) and melanoma (C, D) in
RET-mice at 4 months and 12 months of age, respectively, are presented. Crusts covered the ulcer on the surface inmelanoma (C) but not in the benign melanocytic tumor (A) in RET-mice. Arrowheads indicate a benign melanocytic
tumor (A) and melanoma (C). Hematoxylin and eosin staining was used for histopathological analysis (C, D). Scalebar: 10 µm (D).

We have shown that anchorage-dependent
growth of human melanoma cells was increased
by interaction of c-RET and GDNF after confirming c-RET expression levels in plural
human melanoma cell lines [5]. c-SRC protein,which is expressed in melanoma, is directly
associated with c-RET protein and regulates
the function of c-RET kinase [6]. RFP-RET of thehybrid gene from c-RET and RFP has an oncogenic activity and is constitutively activatedwithout stimulation of ligands [3, 7]. RFP-RETtransgenic mice (RET-mice) carrying RFP-RET
under the control of metallothionein-I promoter
spontaneously develop benign melanocytic
tumors and melanomas without Braf V600E mutation in a stepwise manner [7, 8]. Increased
expression levels of epidermal growth factor
receptor (Egfr) and vascular endothelial growth1062

factor receptor 2 (Vegfr2) in human melanoma
have been reported [9, 10]. These results suggest that RFP-RET, c-RET, Egfr, Vegfr2 and c-Src
molecules are involved in the pathogenesis ofmelanoma.
Attention has recently been paid to medical
applications of non-thermal atmospheric pressure plasmas (NEAPPs) consisting of an ionizedgas. Previous studies showed anti-cancer
effects of NEAPP irradiation in vitro [11-13]. We
have also reported suppressed growth of
benign melanocytic tumors in RET-mice byrepeated NEAPP irradiation via decreased
expression levels of cell cycle regulators andmatrix metalloproteinases (MMPs) [14-16]. In
our previous study, we showed an anti-cancer
effect of single NEAPP irradiation to melano-

Int J Clin Exp Pathol 2016;9(2):1061-1067

Effect of plasma irradiation on expression of tyrosine kinase genes

Figure 2. Non-thermal atmospheric pressure plasma (NEAPP) irradiation. (A, B) Device used for NEAPP irradiation(A) and a bright field of NEAPP irradiation to a melanoma in a RET-mouse (B) are shown. While Ar gas was provided
through a tube, plasma in the main discharge region between two electrodes was excited by applying 10 kV froma 60 Hz commercial power supply. The distance between the two electrodes was 20 mm. Single irradiation for 30
sec was performed on malignant tumors from RET-mice. The RET-mouse was placed on a rubber plate to keep the
distance from the plasma source at 13 mm.

mas in RET-mice via decreased levels of cell
cycle regulators and MMPs [8]. In this study, we
examined the effect of NEAPP irradiation on
expression levels of RFP-RET, c-Ret, Egfr,
Vegfr2 and c-Src in melanomas without
BRAF V600E mutation from RET-mice.
Materials and methods
RET-mice spontaneously developed tumors inthe skin
Benign melanocytic tumors (Figure 1A, 1B) andmelanomas (Figure 1C, 1D) developed spontaneously in our original RET-mice with an intact
immune system [7, 8]. Therefore, RET-mice
could be a strong tool to develop various therapies for melanomas [17, 18]. Experiments using
recombinant DNA were approved by the
Recombination DNA Advisory Committee of
Nagoya University (no. 12-39, 13-59, 13-76).
The animal experiments were approved by the
Animal Care and Use Committee of Nagoya
University (approval no. 27241).
NEAPP irradiation to tumors in RET-mice
Benign melanocytic tumors and melanomas
with or without NEAPP irradiation in RET-micewere used. The NEAPP device used in the present study is shown in Figure 2A. Melanomas
were collected and analyzed 6 hours after single NEAPP irradiation for 30 sec for melanoma

1063

in RET-mice (Figure 2B) following the method
previously described [8].
Quantitative PCR (Q-PCR)
After extracting total RNA from tumors of RETmice, transcript expression levels were measured by the method described previously [14,16]. Sequences of primers used are shown in
Supplementary Table 1.
Results
Diagnosis of tumors in RET-mice
Benign melanocytic tumors and melanomas in
RET-mice were preliminary selected by macroscopic observation (Figure 1A, 1C). After collection of tumors with or without NEAPP irradiation, all of the tumors were histopathologicallydiagnosed by a trained pathologist (Figure 1B,
1D). A benign melanocytic tumor consisted of
uniform cells having round nuclei without mitosis (Figure 1B). Melanoma consisted of atypicalcells having various sizes and shapes of nuclei
with high mitotic activity (Figure 1D).
Effects of single NEAPP irradiation on expression of 5 tyrosine kinases
Transcript expression levels of RFP-RET, c-Ret,
Egfr, Vegfr2 and c-Src in melanomas were 2.0fold, 1.9-fold, 9.7-fold, 1.2-fold and 2.3-foldhigher than those in benign melanocytic

Int J Clin Exp Pathol 2016;9(2):1061-1067

Effect of plasma irradiation on expression of tyrosine kinase genes
Kluppel-like factor 10 (Klf10) in melanomas from RETmice were further examined. Single NEAPP irradiation decreased the expression level of Ybx1 transcriptby 27% (Figure 4A). However, there were comparable expression levels of
p53, Egr1, Gcf2 and Klf10transcripts in melanomas
from RET-mice despite single NEAPP irradiation (Figure 4B-E).
Discussion
In this study, we first confirmed higher expression
levels of RFP-RET, c-Ret,
Egfr, Vegfr2 and c-Src, allof which were previously
reported to be associatedwith melanoma [5, 6, 9,10], suggesting that thekinases are also associated with melanomas in RETmice. Then we demonstrated for the first time that
Figure 3. Transcript expression levels of RFP-RET, c-Ret, Egfr and Vegfr2 in tuexpression levels of c-Ret
mors from RET-mice. (A-E) Relative transcript expression levels (means ± SD)
and Egfr transcripts, butof RFP-RET (A, RFP-RET), c-Ret (B), Epidermal growth factor receptor (C, Egfr),
not those of RFP-RET,
Vascular endothelial growth factor receptor 2 (D, Vegfr2) and c-Src (E) in beVegfr2 and c-Src trannign melanocytic tumors (B, n=6) and melanomas (M, n=5) from RET-mice arescripts, in melanomas fromshown. (F-J) Relative transcript expression levels of RFP-RET (F), c-Ret (G), Egfr
(H), Vegfr2 (I) and c-Src (J) in untreated (Un, n=5) and NEAPP-treated (Tr, n=6)
RET-mice were decreased
melanomas from RET-mice are presented. The transcript expression levels wereby single NEAPP irradiation.
determined by quantitative PCR and normalized with the transcript expression
Our results showed that thelevel of hypoxanthine ribosyltransferase (A-J, Hprt). Statistical significance wasdifference in Egfr expresevaluated by Student’s t-test. **P<0.01, *P<0.05.
sion level between benign
melanocytic tumors andtumors, respectively, in RET-mice (Figure 3A-E).
melanoma is the largest among the 5 kinases.
Moreover, the effects of endogenous c-Ret may
Single NEAPP irradiation decreased expression
be replaced by the effects of the introduced
levels of c-Ret and Egfr transcripts by 70% and
RFP-RET in RET-mice. In fact, kinase activity of
41%, respectively (Figure 3G, 3H). However, thec-Ret was found to be lower than that of RFPexpression levels of RFP-RET, Vegfr2 and c-Src
RET in RET-mice in our previous studies [3, 4,transcripts were comparable in single NEAPP
19]. Together with a previous report showing
irradiated- and unirradiated-melanomas inthat EGFR regulates progression and metastaRET-mice (Figure 3F, 3I, 3J).
sis of human melanoma [20], we focused on
Effects of NEAPP irradiation on expression of
Egfr as a representative molecule that wastranscription factors for Egfr
decreased by single NEAPP irradiation.
Effects of single NEAPP irradiation on transcript
expression levels of Y box protein 1 (Ybx1),
p53, Early growth factor 1 (Egr1), GC-richsequence DNA binding factor 2 (Gcf2) and1064

YBX1, p53 and EGR1 bind to the promoter ofthe EGFR gene and positively control its transcription in human osteosarcoma, breast cancer and colon cancer cells [21-23]. GCF2 andInt J Clin Exp Pathol 2016;9(2):1061-1067

Effect of plasma irradiation on expression of tyrosine kinase genes

Figure 4. Transcript expression levels of Ybx1, p53, Egr1, Gcf2 and Klf10 in
irradiated melanomas from RET-mice. (A-E) Relative transcript expression levels (means ± SD) of Y box protein 1 (A, Ybx1), p53 (B), Early growth factor 1(C, Egr1), GC-rich sequence DNA binding factor 2 (D, Gcf2) and Kluppel-likefactor 10 (E, Klf10) in untreated (Un; n=5) and NEAPP-treated (Tr, n=6) melanomas from RET-mice are presented. Statistical significance was evaluated byStudent’s t-test. **P<0.01.

that single NEAPP irradiation decreases Egfr expression level via decreased
level of Ybx1, a transcription factor promoting expression of Egfr, in melanomas without Braf V600E mutation from RET-mice (Figure 5). Since EGFR could bea clinically potential therapeutic target for melanoma [20], our results suggest that NEAPP irradiation
is a novel option for therapy of melanoma without
BRAF V600E mutation (Figure5).
Acknowledgements

This study was supported
in part by Grants-in-Aid for
Scientific Research (A) (15H01743 and 15H02588) and (B) (24390157), Grant-in-Aid for
Challenging Exploratory Research (26670525),
Grant-in-Aid for Scientific Research on Innovative Areas (24108001) and Grants-in-Aid forYoung Scientists (B) (22770008) from the
Ministry of Education, Culture, Sports, Scienceand Technology (MEXT), the Mitsui & Co., Ltd.
Environment Fund, the Mitsubishi Foundation,
Foundation from Center for Advanced Medical
and Clinical Research Nagoya University
Hospital and NOVARTIS Research Foundation(Japan).

Figure 5. A schematic pathway for regulation of Egfr
expression level. Y box protein 1 (Ybx1), p53 and Early growth response 1 (Egr1) progressively regulate
Egfr expression level. GC-rich sequence DNA binding
factor 2 (Gcf2) and Kluppel-like factor 10 (Klf10) negatively regulate Egfr expression level. Our results suggest that single irradiation of NEAPP decreases Egfr
transcript expression level via suppression of Ybx1
transcript expression. Since EGFR, a regulator formelanoma progression, has been suggested to bea clinical target for melanoma therapy [20], NEAPP
may be useful for melanoma therapy.

Disclosure of conflict of interest
None.
Address correspondence to: Dr. Masashi Kato, Occupational and Environmental Health, Nagoya University Graduate School of Medicine, 65 Tsurumaicho, Showa-ku, Aichi 466-8550, Japan. Tel: +81-52744-2122; Fax: +81-52-744-2122; E-mail: katomsasa@med.nagoya-u.ac.jp

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Effect of plasma irradiation on expression of tyrosine kinase genes
Supplementary Table 1. Sequences of primers
used in quantitative PCR
Gene
RFP-RETRFP-RET
c-Ret
c-Ret
Egfr
Egfr
Vegfr2
Vegfr2
c-Src
c-Src
Ybx1
Ybx1
p53
p53
Egr1
Egr1
Gcf2
Gcf2
Klf10
Klf10
Hprt
Hprt

1

StrandForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseSequence
5’ TCACAGGGGATGCAGTATCT 3’
5’ CCTGGCTCCTCTTCACGTAG 3’
5’ TGACATCAGCAAGGATCTGG 3’
5’ CCCATCGTCATACAGCAGTG 3’
5’ CAAGTGGATGGCTTTGGAAT 3’
5’ TCCATCATAAGGCTTGGACC 3’
5’ TGAAATCTTTGGTGGAAGCC 3’
5’ GGCCTTCCATTTCTGTACCA 3’
5’ TCGTGAGGGAGAGTGAGAC 3’
5’ GCGGGAGGTGATGTAGAAAC 3’
5’ GGTGCAGGAGAGCAAGGTAG 3’
5’ TGCCATCCTCTCTAGGCTGT 3’
5’ TGTCACGCTTCTCCGAAGAC 3’
5’ ATCGTCCATGCAGTGAGGTG 3’
5’ GAGCGAACAACCCTATGAGC 3’
5’ TTTGGCTGGGATAACTCGTC 3’
5’ AACTTCCACAAGTGCCAAGG 3’
5’ GGAAACTTTTCTCGCCACTG 3’
5’ AGCAAGGGTCACTCCTCAGA 3’
5’ TGAAAGGTTTTTCCCCTGTG 3’
5’ TATGTCCCCCGTTGACTGAT 3’
5’ CTTTGCTGACCTGCTGGATT 3’