DOI: 10.1111/exd.12415
Letter to the Editor
www.wileyonlinelibrary.com/journal/EXD
Non-equilibrium atmospheric pressure plasmas modulate cell cycle-related gene expressions in melanocytic tumors of RET-transgenic mice Ichiro Yajima1,2, Machiko Iida2, Mayuko Y. Kumasaka1,2, Yasuhiro Omata2, Nobutaka Ohgami1, Jie Chang2,3, Sahoko Ichihara3, Masaru Hori4 and Masashi Kato1,2 1 Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Aichi, Japan; 2Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; 3 Graduate School of Regional Innovation Studies, Mie University, Tsu, Japan; 4Department of Electrical Engineering and Computer Science, Graduate School of Engineering, Nagoya University, Nagoya, Japan Correspondence: Masashi Kato, Department of 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: +81-52-744-2126, e-mail:
[email protected]
Abstract: The incidence of cutaneous malignant melanoma is
increasing at a greater rate than that of any other cancer in the world. However, an effective therapy for malignant melanoma has not been established. Recently, some studies have shown an antitumor effect of non-equilibrium atmospheric pressure plasmas (NEAPPs) in vitro. Here, we examined the in vivo effect of NEAPP on cell cycle regulators, key elements for malignant transformation, in spontaneously developed benign melanocytic tumors in a hairless animal model. NEAPP irradiation decreased expression levels of cell cycle promoters, Cyclin D1, E1 and E2,
and increased expression level of a cell cycle repressor, p27KIP1. Cyclin D1, E1 and E2 and p27KIP expression levels were associated with malignant transformation of the benign tumor in the animal model. Our results suggest that NEAPP irradiation suppresses malignant transformation of a benign melanocytic tumor via control of the expression levels of cell cycle regulators. Key words: cell cycle – RET – melanoma – non-equilibrium atmospheric pressure plasma
Accepted for publication 15 April 2014
Background
Experimental design
As malignant melanoma is the most serious skin cancer, it is a threat for human life. However, an effective therapy for malignant melanoma has not been established (1–3). In multistep melanomagenesis, one of the mechanisms of melanoma development, human melanoma, develops from a melanocytic naevus. In previous studies in which human tissues were analysed, cell cycle regulators, including Cyclin D1, Cyclin E1, Cyclin E2, Cyclin G1, Pcna, p21Cip1/WAF1 and p27KIP1, have been reported to be associated with melanoma development from melanocytic naevus (2,4–8). We previously developed a hairless mouse line of 304-hr/hr (HL-RET-mice) carrying constitutively activated RET. Benign melanocytic tumors and melanomas spontaneously develop stepwise in HL-RET-mice (9,10). The entire process of tumor development and malignant conversion in HL-RET-mice resembles that of a human melanocytic naevus (9,10). As benign melanocytic tumors develop in HL-RET-mice without exception (10), HL-RET-mice could be a strong tool to develop therapies preventing development of melanoma by targeting benign tumors. Recently, non-equilibrium atmospheric pressure plasmas (NEAPPs) have gained attention in the field of clinical applications (11). Previous studies have showed that the plasma irradiation is effective for melanoma therapy in vitro (12–14). To our knowledge, however, information about melanoma therapy by NEAPPs in vivo has not been presented.
Previously established HL-RET-mice (10) were used. The Animal Care and Use Committee (approval no. 2510052 and 26317) and Recombination DNA Advisory Committee (approval no. 12-04 and 12-59) in Chubu University and Nagoya University approved this study. Materials and methods of the NEAPP treatment have been previously reported (15) (Figure S1). The method of treatment was arranged to the treatment for mice. Briefly, the flow rate of Ar gas was set at 2 slm (standard litre/min), and the distance between the plasma source and the tumor surface was fixed at 13 mm. Tumor size at the beginning of treatment was adjusted to 100 cm3. While Ar gas was flowing, plasma in the main discharge region was excited by applying 10 kV from a 60Hz commercial power supply to two electrodes that were 20 mm apart. The plasma or Ar gas for the control was exposed to a 7 cm2 area including the tumor surface for 1 min at once per week, and after 12 weeks, tumors were extirpated and total RNAs were purified. Total RNAs were purified from tumors using a High Pure RNA purification Kit (Roche) according to the manufacturer’s protocol. Skin regions were removed from specimens, and then, RNAs were purified from tumors. cDNA was then synthesized by reverse transcription of total RNA using Superscript III reverse transcriptase included in the RT enzyme mix and RT reaction mix (Invitrogen) according to the manufacturer’s protocol. Q-PCR with SYBR green was performed using FastStart Universal SYBR Green Master (Roche) in a Thermal Cycler Dice Real Time System (TaKaRa). The expression levels of murine CyclinD1, CyclinE1, CyclinE2, CyclinG1, Pcna, p21 Cip1/WAF1 and p27KIP1 mRNA measured by Q-PCR were adjusted through the expression level of murine Hprt mRNA. Sequences of primers for these genes are described in Table S1.
Questions addressed The aim of this work was the validation of NEAPP irradiationmediated effects in melanocytic tumors that spontaneously developed in HL-RET-mice with focus on cell cycle regulators in vivo.
424
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 424–448
Letter to the Editor
(a)
(a)
(b)
(b)
Figure 1. Expression levels of cell cycle-associated genes in melanocytic tumors. (a) Expression levels of cell cycle-associated genes in untreated and NEAPP-treated tumors from HL-RET-mice. Expression levels of Cyclin D1, Cyclin E1, Cyclin E2, Cyclin G1, Pcna, p21Cip1/WAF1 and p27KIP1 mRNAs evaluated by Q-PCR in untreated and NEAPP-treated benign melanocytic tumors from HL-RET-mice. The results are presented as ratios (mean SD; n = 5) of each mRNA relative to NEAPP-untreated tumors. As a control, Ar gas was applied at the same flow rate [2 slm (standard litre/min)]. (b) Expression levels of cell cycle-associated genes in benign melanocytic tumors (B) and melanomas (M) from HL-RET-mice. Expression levels of Cyclin D1, Cyclin E1, Cyclin E2, Cyclin G1, Pcna, p21Cip1/WAF1 and p27KIP1 mRNAs evaluated by Q-PCR in benign melanocytic tumors and melanomas from HL-RET-mice. The results are presented as ratios (mean SD; five specimens) of each mRNA relative to benign melanocytic tumors. Significantly different (*, P < 0.05; **, P < 0.01) by Student’s t-test.
Results and Discussion We first examined expression levels of cell cycle-associated genes, Cyclin D1, Cyclin E1, Cyclin E2, Cyclin G1, Pcna, p21Cip1/WAF1 and p27KIP1, in untreated and plasma-treated benign melanocytic tumors of HL-RET-mice by quantitative polymerase chain reaction (Q-PCR) analysis (Fig. 1a). Expression levels of Cyclin D1, E1 and E2 genes, such as cell cycle mediators (2,4,6), were significantly decreased in plasma-treated tumors, whereas expression level of p27KIP1, a cell cycle repressor (8), was significantly increased in plasma-treated tumors. To determine the biological significance of the NEAPP treatment-mediated modulation of cell cycle-associated gene expression, we then compared gene expression levels in benign tumors and melanomas of HL-RET-mice by Q-PCR analysis (Fig. 1b). Expression levels of all cell cycle mediators were increased in melanomas, whereas expression level of the cell cycle repressor p27 KIP1 was significantly decreased in melanomas. Results shown in Fig. 1b suggest that up-regulation of Cyclin D1, E1 and E2 expression and down-regulation of Pcna and p27KIP1 expression are associated with the development of melanoma. Tumor size was measured in plasma-treated or non-treated tumors to elucidate the relationship between modulation of cell cycle-associated gene expression and tumor growth (Fig. 2). Growth ratio of
References 1 Chin L, Garraway L A, Fisher D E. Genes Dev 2006: 20: 2149–2182. 2 Yajima I, Kumasaka M Y, Thang N D et al. J Skin Cancer 2011: 2011: 730170. 3 Yajima I, Kumasaka M Y, Thang N D et al. Dermatol Res Pract 2012: 2012: 354191. 4 Bales E, Mills L, Milam N et al. Cancer Res 2005: 65: 692–697. 5 Ben-Izhak O, Bar-Chana M, Sussman L et al. Histopathology 2002: 41: 519–525. 6 Carreira S, Goodall J, Denat L et al. Genes Dev 2006: 20: 3426–3439.
Figure 2. Effects of NEAPP treatment to benign melanocytic tumors. (a) Ratio of tumor size in untreated and NEAPP-treated benign melanocytic tumors from HL-RET-mice. The results are presented as ratios (mean SD; n = 5) of each tumor size (12W/0W) relative to NEAPP-untreated tumors. (b) Haematoxylin and eosin staining of sections from untreated and NEAPP-treated benign melanocytic tumors.
plasma-treated tumors was significantly reduced compared with that of non-treated tumors (Fig. 2a), and no effect of Ar gas for non-plasma treatment was found in tumor sections (Fig. 2b). Results shown in Fig. 2 suggest that plasma treatment of benign tumors represses tumor growth. Taken together, our results suggest that NEAPP treatment for melanocytic benign tumors suppresses the development of melanoma by regulation of cell cycle mediators in HL-RET-mice. NEAPP treatment might be a useful tool for preventing the development of melanoma from melanocytic nevi.
Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research (B) (No. 24390157 and 24406002) and (C) (No. 25340052, 25461717), Grant-inAid for Challenging Exploratory Research (No. 23650241, 26670525), Grantin-Aid for Restart Postdoctoral Fellowship (No. 25-40080), Grant-in-Aid for Scientific Research on Innovative Areas (No. 24108001) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT); the Naito Foundation Natural Science Scholarship; the Uehara Memorial Foundation and The Cosmetology Research Foundation.
Author contributions MK provided HL-RET-mice. HM provided the instrument for plasma irradiation. IY, MK and MH designed the research study. IY, MI, MYK, YO and JC performed experiments, analysed data, prepared the Figures and Table and drafted the manuscript under the supervision of NO, SI, MH and MK. IY and MK wrote the paper.
Conflict of interests The authors have declared no conflicting interests.
7 Horne M C, Goolsby G L, Donaldson K L et al. J Biol Chem 1996: 271: 6050–6061. 8 Ibanez I L, Bracalente C, Notcovich C et al. PLoS ONE 2012: 7: e44502. 9 Kato M, Takahashi M, Akhand A A et al. Oncogene 1998: 17: 1885–1888. 10 Thang N D, Yajima I, Nakagawa K et al. J Dermatol Sci 2012: 65: 207–212. 11 Haertel B, Wende K, von Woedtke T et al. Exp Dermatol 2011: 20: 282–284. 12 Arndt S, Wacker E, Li Y F et al. Exp Dermatol 2013: 22: 284–289.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2014, 23, 424–448
13 Lupu A R, Georgescu N, Calugaru A et al. Roum Arch Microbiol Immunol 2009: 68: 136–144. 14 Sensenig R, Kalghatgi S, Cerchar E et al. Ann Biomed Eng 2011: 39: 674–687. 15 Iseki S, Nakamura K, Hayashi M et al. Appl Phys Lett 2012: 100: 113702.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Scheme of generation of non-equilibrium atmospheric pressure plasma (NEAPP). Table S1. Primer sequences of genes for PCR.
425
