Int J Clin Exp Pathol 2015;8(8):9326-9331
www.ijcep.com /ISSN:1936-2625/IJCEP0011212

Original Article
Decreased expression levels of cell cycle regulatorsand matrix metalloproteinases in melanoma from
RET-transgenic mice by single irradiation of
non-equilibrium atmospheric pressure plasmas
Machiko Iida1,2*, Yasuhiro Omata1*, Chihiro Nakano1, Ichiro Yajima1,2, Toyonori Tsuzuki3, Kenji Ishikawa4,
Masaru Hori4, Masashi Kato1,2
Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine,
65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan; 2Department of Biomedical Sciences, Unit ofEnvironmental Health Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Aichi 487-8501,
Japan; 3Department of Pathology, Nagoya Daini Red Cross Hospital, Showa-ku, Nagoya, Aichi 466-8650, Japan;
4
Department of Electrical Engineering and Computer Science, Graduate School of Engineering, Nagoya University,Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan. *Equal contributors.
1

Received June 9, 2015; Accepted July 24, 2015; Epub August 1, 2015; Published August 15, 2015
Abstract: Since effective therapies for melanoma with BRAFV600E mutation are being developed, interest has beenshown in the development of therapies for melanoma without BRAFV600E mutation. Recently, interest has also beenshown in medical application of non-nequilibrium atmospheric pressure plasmas (NEAPPs). We previously suggested that repeated NEAPP irradiation to spontaneously developed benign melanocytic tumors in RFP-RET-transgenic
mice (RET-mice) not only suppresses tumor growth but also prevents malignant transformation. In this study, wefirst confirmed that transcript expression levels of tumor growth regulators (CyclinD1, D2, E1, E2, G2 and PCNA but
not CyclinG1) and tumor invasion regulators [Matrix metalloproteinase (MMP)-2, -9 and -14 and melanoma celladhesion molecule (MCAM)] in melanomas were significantly higher than those in benign melanocytic tumors inRET-mice. We then showed that transcript expression levels of CyclinE1, G1 and G2 and MMP-2 and -9 in melanomas from RET-mice were significantly decreased by single NEAPP irradiation, whereas transcript expression levels ofCyclinD1, D2, E2, PCNA, MCAM and MMP-14 were comparable in untreated and NEAPP-treated melanomas. Since
no BrafV600E mutation melanomas have been found in RET-mice, our results suggest that single NEAPP irradiation isa potential therapeutic tool for melanoma without BRAFV600E mutation through modulation of the expression levelsof tumor growth and invasion regulators.
Keywords: Melanoma, non-equilibrium atmospheric pressure plasmas, tumor growth, invasion

Introduction
Melanoma, the most aggressive skin cancer,
is rapidly increasing worldwide because ofincreased ultraviolet irradiation caused bydepletion of the ozone layer. Generally, melanoma has been reported to be resistant tovarious therapies including X-ray irradiation.
Recently, interest has been shown in a molecular target therapy for BRAF V600E mutation as a
new therapy for melanoma [1]. However, therapy for melanoma without BRAF V600E mutation
remains difficult. Since the results of a previous
study showed that about half of melanoma

cases have no mutation of BRAF V600E [1, 2],development of a novel therapy for melanoma
without BRAF V600E mutation is one of the key
elements in melanoma research.
Interest has recently been shown in applicationof non-nequilibrium atmospheric pressure plasmas (NEAPPs) for the medical field including
cancer therapy. In fact, there are some reports
showing anti-cancer effects of NEAPPs for various cancers including melanoma in vitro [3-5].
However, studies showing an in vivo effect ofNEAPPs on transplanted cancer in animals arelimited [6], and there are even fewer studies anPlasma irradiation to melanoma in RET-miceanoma from benign melanocytic tumors in RET-mice[12]. Matrix metalloproteinase (MMP)-2 and -9 weresuggested to be associatedwith tumor growth [9, 13]
and malignant transformation [9, 14, 15] in addition
to tumor invasion andmetastasis [9, 15] intumors in RET-mice. MMP14 and melanoma celladhesion molecule (MCAM)
have been reported to be
regulators of the activity
and expression of MMP-2,
respectively [16, 17] andhave been suggested to beassociated with invasion ofmelanoma from RET-mice[9, 18]. Our previous study
also suggested that MCAM
and MMPs are involved
Figure 1. Single non-nequilibrium atmospheric pressure plasma (NEAPP) irrain melanoma development
diation to melanomas. (A, B) A schema (A) and a photograph with a dark fieldfrom benign melanocytic(B) of NEAPP irradiation to melanomas in RET-mice are presented. (C) Temperatumors in RET-mice [15,tures (means ± SD) measured by an infrared thermometer (CA380; CASON,Inc) on the melanoma surface before (n=6) and just after (n=6) single NEAPP
18]. Since repeated NEAPP
irradiation are shown. Statistical significance was evaluated by the paired Stuirradiation for benign meladent’s t-test. (D) HE-stained microscopic appearances of melanomas in RETnocytic tumors in RET-mice
mice before (Untreated) and 6 hours after (Treated) single NEAPP irradiation.
for 11 weeks decreasedtranscript expression levelsof Cyclins, MCAM and MMPs in our previous
in vivo effect of NEAPPs on spontaneously
study, we proposed that NEAPP irradiation
developed cancer in animals and humans.
might suppress malignant transformation ofMultistep melanomagenesis, melanoma thatbenign melanocytic tumors in RET-mice [12,has developed from a melanocytic nevus, has15, 18]. Based on our previous results, webeen reported in patients with large congenital
examined the effect of NEAPP irradiation on
melanocytic nevi [7]. We previously developed
cutaneous tumors after transformation (melanoma) in RET-mice in this study.
RFP-RET-transgenic mice (RET-mice) under
the control of the metallothionein-I promoter/Materials and methods
enhancer, in which benign melanocytic tumors
and melanoma spontaneously develop with anTumors
intact immune system [8]. The entire processof melanomagenesis in RET-mice resembles
Benign melanocytic tumors and melanomas
that of a human melanocytic nevus [9, 10].
in our original RET-mice [8] were used after
Since macroscopic and microscopic appearconfirming no Braf V600E mutation in melanomas
ances of melanoma used in RET-mice are comby DNA sequence analysis according to theparable with those of melanoma in humans,
method described previously [19].
RET-mice are used worldwide as a standard
animal model for melanoma [8-11].
NEAPP irradiation
Previous studies showed that cell cycle promoters such as CyclinD1, E1 and E2 and PCNA arepotentially involved in the development of mel-9327

Transcript expression levels of cell cycle
promoters, MCAM and MMPs at 6 hours after
single NEAPP irradiation for 30 seconds wereInt J Clin Exp Pathol 2015;8(8):9326-9331

Plasma irradiation to melanoma in RET-miceFigure 2. Transcript expression levels of CyclinD1, CyclinD2, CyclinE1, CyclinE2, CyclinG1, CyclinG2, and PCNA intumors from RET-mice. (A-G) Relative transcript expression levels (means ± SD) of CyclinD1 (A), CyclinD2 (B), CyclinE1 (C), CyclinE2 (D), CyclinG1 (E), CyclinG2 (F), and PCNA (G) in benign melanocytic tumors (Benign, n=6) andmelanoma (Malignant, n=6) of RET-mice are presented. (H-N) Relative transcript expression levels (means ± SD) ofCyclinD1 (H), CyclinD2 (I), CyclinE1 (J), CyclinE2 (K), CyclinG1 (L), CyclinG2 (M), and PCNA (N) in NEAPP-irradiated(n=6) and unirradiated (n=5) melanomas from RET-mice are presented. The transcript expression levels were determined by quantitative PCR and normalized with the transcript expression level of hypoxanthine ribosyltransferase(Hprt) (A-N). Statistical significance was evaluated by the Mann-Whitney U test. *p<0.05, **p<0.01.

examined in pathologically diagnosed benign
melanocytic tumors and melanomas from RETmice. The method used for NEAPP irradiation(Figure 1A and 1B) in this study was the sameas our previous method [12] except for thedifference of single or repeated irradiation.
Quantitative PCR (Q-PCR)
Q-PCR was performed according to the method
previously described [12, 15, 18].

9328

Approval
The experiments using recombinant DNA wereapproved by the Recombination DNA Advisory
Committee of Nagoya University (approval No.
12-59, 13-59, 13-76) and Chubu University(approval No. 12-04). The animal experiments
were approved by the Animal Care and Use
Committee of Nagoya University (approval No.
27241) and Chubu University (approval No.
2710048).

Int J Clin Exp Pathol 2015;8(8):9326-9331

Plasma irradiation to melanoma in RET-mice
Effect of NEAPP irradiation
on expression of regulatorsof the cell cycle
Constitutive expression levels of cell cycle regulatory
genes, CyclinD1, D2, E1, E2,G1, G2 and PCNA, in benign
tumors and melanomas
from RET-mice are shown in
Figure 2A-G. Transcript expression levels of CyclinD1,E1, E2 and PCNA, but notthat of CyclinG1, in melanomas were significantly
higher than those in benign
melanocytic tumors, as wasobserved in our previous
study [12]. Increased transcript expression levels ofCyclinD2 and G2 in melanomas from RET-mice compared to those in benign
melanocytic tumors were
newly shown in this study.

Figure 3. Transcript expression levels of MCAM and MMPs in melanomas fromRET-mice. (A-D) Relative transcript expression levels (means ± SD) of MCAM
(A), MMP-2 (B), MMP-9 (C) and MMP-14 (D) in melanocytic benign tumors(Benign; n=6) and melanomas (Malignant; n=6) from RET-mice. (E-H) Relativetranscript expression levels (means ± SD) of MCAM (E), MMP-2 (F), MMP-9 (G)and MMP-14 (H) in untreated (Untreated; n=6) and NEAPP-treated (Treated;n=6) melanomas from RET-mice are presented. Statistical significance wasanalyzed by the Mann-Whitney U test. *P<0.05; **P<0.01.

Results
Effects of NEAPP irradiation on temperatureand morphology of melanoma
The temperatures before and just after single
NEAPP irradiation on the melanoma surface
were comparable (Figure 1C). Histopathologicalanalysis showed comparable microscopic
appearances of untreated and NEAPP-treatedmelanoma at 6 hours after irradiation (Figure1D).

9329

Transcript expression levelsof CyclinE1, G1 and G2 inmelanoma from RET-micewere significantly decreased
by single NEAPP irradiation(Figure 2J, 2L, 2M). Therewas no statistical differencein CyclinD1, D2, E2 andPCNA transcript expressionlevels between untreatedand NEAPP-treated melanomas (Figure 2H, 2I, 2K, 2N).
Effect of NEAPP irradiation
on expression of regulatorsof invasion

Constitutive transcript expression levels ofinvasion regulatory genes, MCAM and MMP-2,-9 and -14, in benign melanocytic tumors andmelanomas from RET-mice are shown in Figure
3A-D. Transcript expression levels of all of themolecules in melanomas were significantly
higher than those in benign melanocytic tumors, as was observed in our previous studies[15, 18].
Transcript expression levels of MMP-2 and -9 inmelanomas from RET-mice were significantly

Int J Clin Exp Pathol 2015;8(8):9326-9331

Plasma irradiation to melanoma in RET-micedecreased by single NEAPP irradiation (Figure2F and 2G). There was no statistical differencein MCAM and MMP-14 transcript expression
levels between untreated and NEAPP-treatedmelanomas (Figure 2E and 2H).
Discussion
We demonstrated for the first time that single
NEAPP irradiation decreases expression levelsof cell cycle regulators (Cyclin E1, G1 and G2)
and invasion regulators (MMP-2 and -9) inmelanoma from RET-mice with limited effects
on temperature and histopathological appearance.
After repeated NEAPP irradiation to benign
melanocytic tumors in original RET-mice for 11weeks, the effect was evaluated 12 weeks
after the first irradiation in our previous studies[12, 15, 18]. However, survival for 12 weeks
after the first irradiation might not be easy for
RET-mice with melanoma, and we therefore
chose single NEAPP irradiation to melanoma in
RET-mice as our first trial. To our knowledge,
this is the first study showing the effect ofsingle NEAPP irradiation on spontaneously
developed melanoma in mice with an intact
immune system.
We showed decreased expression levels ofCyclinE1, G1 and G2 in NEAPP-treated melanomas after showing increased expression levelsof CyclinD, E and G in melanomas from RETmice. Since cell cycle promoters such as CyclinE
and G have been suggested to be involved in
the growth of tumors including melanoma inhumans and mice [12, 20], our results suggest
a potentially suppressive effect of single NEAPP
irradiation on melanoma growth. We furthershowed decreased expression levels of MMP-2
and -9 in NEAPP-treated melanomas after
showing increased expression levels of MMP-2
and -9 in the melanomas from RET-mice. Since
the major role of MMP-2, -9 and -14 and MCAM
has been reported to be regulation of the invasion of various cancers including melanoma inhumans and mice [9, 21], our results suggest apotentially inhibitory effect of single NEAPP
irradiation on melanoma invasion.

Moreover, not only temperature but also microscopic appearance of melanoma was comparable before and after NEAPP irradiation. Theseresults suggest that NEAPP irradiation-mediated decrease in the expression levels of regulatory molecules for tumor growth and invasion
may be independent of NEAPP irradiationmediated tissue damage.
Our results suggest that NEAPP irradiation is anovel candidate for therapy of melanoma without BRAF V600E mutation because there were nomelanomas with Braf V600E mutation in RETmice. Our results are encouraging for further
studies aimed at clinical application of NEAPPirradiation for melanoma therapy.
Acknowledgements
This work was supported in part by Grants-inAid for Scientific Research (A) (No. 15H01743
and 15H02588), (B) (No. 24390157 and24406002) and (C) (No. 25340052 and25461717), Grant-in-Aid for Challenging
Exploratory Research (No. 26670525), Grantin-Aid for Scientific Research on Innovative
Areas (No. 24108001) from the Ministry of
Education, Culture, Sports, Science andTechnology (MEXT) and Toyoaki ScholarshipFoundation, the Mitsui & Co., Ltd. Environment
Fund and Foundation from Center for Advanced
Medical and Clinical Research Nagoya
University Hospital.
Disclosure of conflict of interest
None.
Address correspondence to: Dr. Masashi Kato,
Occupational and Environmental Health, Nagoya
University Graduate School of Medicine, 65
Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550,
Japan. Tel: +81-52-744-2122; Fax: +81-52-7442126; E-mail: katomasa@med.nagoya-u.ac.jp

References[1]

Expression levels of CyclinD1, D2 and E2,PCNA, MCAM and MMP-14 were comparable
in untreated and NEAPP-treated melanomas.

Lito P, Rosen N, Solit DB. Tumor adaptation
and resistance to RAF inhibitors. Nat Med
2013; 19: 1401-1409.
Curtin JA, Fridlyand J, Kageshita T, Patel HN,Busam KJ, Kutzner H, Cho KH, Aiba S, Bröcker
EB, LeBoit PE, Pinkel D, Bastian BC. Distinct
sets of genetic alterations in melanoma. NEngl J Med 2005; 353: 2135-2147.

9330

Int J Clin Exp Pathol 2015;8(8):9326-9331[2]

Plasma irradiation to melanoma in RET-mice[3]

Fridman G, Shereshevsky A, Jost MM, Brooks
AD, Fridman A, Gutsol A, Vasilets V, Friedman
G. Floating electrode dielectric barrier discharge plasma in air promoting apoptotic behavior in melanoma skin cancer cell lines.
Plasma Chemistry and Plasma Processing2007; 27: 163-176.
[4] Tanaka H, Mizuno M, Ishikawa K, Nakamura K,Kajiyama H, Kan H, Kikkawa F, Hori M. Plasmaactivated medium selectively kills glioblastoma brain tumor cells by downregulating a survival signaling molecule, AKT kinase. Plasma
Med 2011; 1: 265-277.
[5] Iseki S, Nakamura K, Hayashi M, Tanaka H,
Kondo H, Kajiyama H, Kano H, Kikkawa F, Hori
M. Selective killing of ovarian cancer cells
through induction of apoptosis by nonequilibrium atmospheric pressure plasma. Appl Phys
Lett 2012; 100: 113702.
[6] Utsumi F, Kajiyama H, Nakamura K, Tanaka H,Mizuno M, Ishikawa K, Kondo H, Kano H, Hori
M, Kikkawa F. Effect of indirect nonequilibrium
atmospheric pressure plasma on anti-proliferative activity against chronic chemo-resistantovarian cancer cells in vitro and in vivo. PLoS
One 2013; 8: e81576.
[7] Marghoob AA. Congenital melanocytic nevi.
Evaluation and management. Dermatol Clin
2002; 20: 607-616.
[8] Kato M, Takeda K, Kawamoto Y, Tsuzuki T,Hossain K, Tamakoshi A, Kunisada T,Kambayashi Y, Ogino K, Suzuki H, Takahashi
M, Nakashima I. c-Kit-targeting immunotherapy for hereditary melanoma in a mouse model.
Cancer Res 2004; 64: 801-806.
[9] Kato M, Takahashi M, Akhand AA, Liu W, Dai Y,
Shimizu S, Iwamoto T, Suzuki H, Nakashima I.
Transgenic mouse model for skin malignant
melanoma. Oncogene 1998; 17: 1885-1888.
[10] Thang ND, Yajima I, Nakagawa K, Tsuzuki T,
Kumasaka MY, Ohgami N, Ly TB, Iwamoto T,Watanabe D, Kato M. A novel hairless mousemodel for malignant melanoma. J Dermatol Sci
2012; 65: 207-212.
[11] Végran F, Berger H, Boidot R, Mignot G,Bruchard M, Dosset M, Chalmin F, Rébé C,Dérangère V, Ryffel B, Kato M, Prévost-Blondel
A, Ghiringhelli F, Apetoh L. The transcription
factor IRF1 dictates the IL-21-dependent anticancer functions of TH9 cells. Nat Immunol
2014; 15: 758-766.

9331[12] Yajima I, Iida M, Kumasaka MY, Omata Y,Ohgami N, Chang J, Ichihara S, Hori M, Kato M.
Non-equilibrium atmospheric pressure plasmas modulate cell cycle-related gene expressions in melanocytic tumors of RET-transgenic
mice. Exp Dermatol 2014; 23: 424-425.
[13] Shi H, Liu L, Liu LM, Geng J, Chen L. Inhibitionof tumor growth by β-elemene through downregulation of the expression of uPA, uPAR,
MMP-2, and MMP-9 in a murine intraocularmelanoma model. Melanoma Res 2015; 25:15-21.
[14] Coussens LM, Werb Z. Matrix metalloproteinases and the development of cancer. Chem
Biol 1996; 3: 895-904.
[15] Iida M, Yajima I, Ohgami N, Tamura H, Takeda
K, Ichihara S, Hori M, Kato M. The effects ofnon-thermal atmospheric pressure plasma irradiation on expression levels of matrix metalloproteinases in benign melanocytic tumors inRET-transgenic mice. Eur J Dermatol 2014; 24:392-394.
[16] Zigler M, Villares GJ, Dobroff AS, Wang H,Huang L, Braeuer RR, Kamiya T, Melnikova VO,Song R, Friedman R, Alani RM, Bar-Eli M.
Expression of Id-1 is regulated by MCAM/
MUC18: a missing link in melanoma progression. Cancer Res 2011; 71: 3494-3504.
[17] Overall CM, López-Otín C. Strategies for MMP
inhibition in cancer: innovations for the posttrial era. Nat Rev Cancer 2002; 2: 657-672.
[18] Omata Y, Iida M, Yajima I, Takeda K, Ohgami N,Hori M, Kato M. Non-thermal atmosphericpressure plasmas as a novel candidate for preventive therapy of melanoma. Environ HealthPrev Med 2014; 19: 367-369.
[19] Heinzerling L, Kühnapfel S, Meckbach D,
Baiter M, Kaempgen E, Keikavoussi P, Schuler
G, Agaimy A, Bauer J, Hartmann A, Kiesewetter
F, Schneider-Stock R. Rare BRAF mutations inmelanoma patients: implications for molecular
testing in clinical practice. Br J Cancer 2013;
108: 2164-2171.
[20] Georgieva J, Sinha P, Schadendorf D.
Expression of cyclins and cyclin dependent
kinases in human benign and malignant
melanocytic lesions. J Clin Pathol 2001; 54:
229-235.
[21] Dye DE, Medic S, Ziman M, Coombe DR.
Melanoma biomolecules: independently identified but functionally intertwined. Front Oncol
2013; 3: 252.

Int J Clin Exp Pathol 2015;8(8):9326-9331