One microgram of genomic DNA sample was sodium bisulfite-converted using the EZ DNA Methylation Kit (Zymo Research). (lung) (B) cancer cells were treated with plasma for 10 times (30 sec each time with an hour interval between exposures), and apoptosis was analyzed by FACS. The assay was performed in triplicate and the result is given by a representative FACS diagram. The ratio of cells undergoing apoptosis is usually denoted by a bar graph with average and standard errors.(DOCX) pone.0129931.s003.docx (122K) GUID:?622FFE38-6A2F-4BCE-9B31-F5C750D276E0 S4 Fig: Anti-proliferation effect of plasma around the breast cancer cells. MCF-7 (A) and MDA-MB-231 cells (B) were treated with plasma and the cell proliferation assay was carried out through colony forming assay. The top and bottom row of each panel are results for plasma non-treated and treated cells, respectively.(DOCX) pone.0129931.s004.docx (907K) GUID:?DB838DBA-58B2-443C-8FA9-14589AB90901 S1 Table: Sequences of primers employed in this study. (DOCX) pone.0129931.s005.docx (13K) GUID:?3C66BDB8-E5BF-4B28-A15B-EEC3BE3F3D51 Data Availability StatementAll relevant data are available from the NCBI Gene Expression Omnibus (GEO) ( Abstract Cold atmospheric plasma (plasma) has emerged as a novel tool for a cancer treatment option, having been successfully applied to a few types of cancer cells, as well as tissues. However, to date, no studies have been performed to examine the Protopanaxatriol effect of plasma on epigenetic alterations, including CpG methylation. In this study, the effects of plasma on DNA Protopanaxatriol methylation changes in breast cancer cells were examined by treating cultured MCF-7 and MDA-MB-231 cells, representing estrogen-positive and estrogen-negative cancer cells, respectively, with plasma. A pyrosequencing analysis of indicated that a specific CpG site was induced to be hypomethylated from 23.4 to 20.3% (p < 0.05) by plasma treatment in the estrogen-negative MDA-MB-231 cells only. A genome-wide methylation analysis identified cellular movement, connective tissue development and function, tissue development and cell-to-cell signaling and conversation, cell death and survival, cellular development as the top networks. Of the two cell types, the MDA-MB-231 cells underwent a higher rate of apoptosis and a decreased proliferation rate upon plasma treatment. Taken together, these results indicate that plasma induces epigenetic and cellular changes in a cell type-specific manner, suggesting that a careful screening of target cells and tissues is necessary for the potential application of plasma as a cancer treatment option. Introduction Non-thermal atmospheric pressure plasma is usually ionized media that contains a mixture of active particles, including electrons, ions, free radicals, reactive molecules and photons [1, 2]. Part of this mixture consists of reactive oxygen and nitrogen species, such as ozone, superoxide, hydroxyl radicals, singlet oxygen, atomic oxygen, nitric oxide, nitrogen dioxide, nitrite, and nitrates [3, 4]. Plasma has recently emerged in multiple medical applications, having been shown to be highly effective in wound healing and blood coagulation, as well as in the treatment of various diseases, including cancer [5, 6]. For example, in ovarian cancer, chronic chemo-resistant ovarian cancer cells in plasma-activated medium showed decreased cell viability. Furthermore, in a murine subcutaneous tumor-formation model, the injection of plasma-activated media resulted in an inhibition of the ovarian cancer cell-inoculated tumor [7]. In head and neck carcinomas, cold plasma selectively impaired cancer cell lines through non-apoptotic mechanisms, while having a minimal effect on normal oral cavity epithelial cell lines [8]. In breast cancer, it was demonstrated that plasma modified for Protopanaxatriol specific conditions selectively ablated Rabbit Polyclonal to OR2T2 metastatic breast cancer cells by inhibiting the migration and invasion of the cells, while minimally influencing healthy bone marrow mesenchymal stem cells [1]. In addition, cold plasma has been proven to be effective in various other cancer types, including glioma [9], melanoma [10], and pancreatic cancer [11], inducing apoptosis and/or senescence of the cancer cells. The molecular mechanisms underlying the changes in cellular activity by plasma have been explained in a few cases. For example, cold plasma-treated oral cavity squamous cell carcinoma was arrested at the sub-G1 phase and the arrest was associated with DNA damage and the ATM/p53 signaling pathway in SCC25 cells [12]..