Non-thermal atmospheric pressure plasma (NTAPP) is an ionized gas at room temperature and has potential as a new apoptosis-promoting cancer therapy that acts by generating reactive oxygen species (ROS). However, it is imperative to determine its selectivity and standardize the components and composition of NTAPP. Here, we designed an NTAPP-generating apparatus combined with a He gas feeding system and demonstrated its high selectivity toward p53-mutated cancer cells. We first determined the proper conditions for NTAPP exposure to selectively induce apoptosis in cancer cells. The apoptotic effect of NTAPP was greater for p53-mutated cancer cells; artificial p53 expression in p53-negative HT29 cells decreased the pro-apoptotic effect of NTAPP. We also examined extra- and intracellular ROS levels in NTAPP-treated cells to deduce the mechanism of NTAPP action. While NTAPP-mediated increases in extracellular nitric oxide (NO) did not affect cell viability, intracellular ROS increased under NTAPP exposure and induced apoptotic cell death. This effect was dose-dependently reduced following treatment with ROS scavengers. NTAPP induced apoptosis even in doxorubicin-resistant cancer cell lines, demonstrating the feasibility of NTAPP as a potent cancer therapy. Collectively, these results strongly support the potential of NTAPP as a selective anticancer treatment, especially for p53-mutated cancer cells.

Non-thermal atmospheric pressure plasma (NTAPP) is defined as a partially ionized gas with electrically charged particles at atmospheric pressure. Our study showed that exposure to NTAPP generated in a helium-based dielectric barrier discharge (DBD) device increased the proliferation of adipose tissue-derived stem cells (ASCs) by 1.57-fold on an average, compared with untreated cells at 72 h after initial NTAPP exposure. NTAPP-exposed ASCs maintained their stemness, capability to differentiate into adipocytes but did not show cellular senescence. Therefore, we suggested that NTAPP can be used to increase the proliferation of ASCs without affecting their stem cell properties. When ASCs were exposed to NTAPP in the presence of a nitric oxide (NO) scavenger, the proliferation-enhancing effect of NTAPP was not obvious. Meanwhile, the proliferation of NTAPP-exposed ASCs was not much changed in the presence of scavengers for reactive oxygen species (ROS). Also, Akt, ERK1/2, and NF-κB were activated in ASCs after NTAPP exposure. These results demonstrated that NO rather than ROS is responsible for the enhanced proliferation of ASCs following NTAPP exposure. Taken together, this study suggests that NTAPP would be an efficient tool for use in the medical application of ASCs both in vitro and in vivo.

Previously, we showed that exposure of human normal and cancer cells to a 6 mT, 60 Hz gradient electromagnetic field (EMF) induced genotoxicity. Here, we investigated the cellular effects of a uniform EMF. Single or repetitive exposure to a 6 mT, 60 Hz uniform EMF neither induced DNA damage nor affected cell viability in HeLa and primary IMR-90 fibroblasts. However, continuous exposure of these cells to an EMF promoted cell proliferation. Cell viability increased 24.4% for HeLa and 15.2% for IMR-90 cells after a total 168 h exposure by subculture. This increase in cell proliferation was directly correlated with EMF strength and exposure time. When further incubated without EMF, cell proliferation slowed down to that of unexposed cells, suggesting that the proliferative effect is reversible. The expression of cell cycle markers increased in cells continuously exposed to an EMF as expected, but the distribution of cells in each stage of the cell cycle did not change. Notably, intracellular reactive oxygen species levels decreased and phosphorylation of Akt and Erk1/2 increased in cells exposed to an EMF, suggesting that reduced levels of intracellular reactive oxygen species play a role in increased proliferation. These results demonstrate that EMF uniformity at an extremely low frequency (ELF) is an important factor in the cellular effects of ELF-EMF.

Due to the rapid development of mobile phone technology, we are continuously exposed to 1.7 GHz LTE radio frequency electromagnetic fields (RF-EMFs), but their biological effects have not been clarified. Here, we investigated the non-thermal cellular effects of these RF-EMFs on human cells, including human adipose tissue-derived stem cells (ASCs), Huh7 and Hep3B liver cancer stem cells (CSCs), HeLa and SH-SY5Y cancer cells, and normal fibroblast IMR-90 cells. When continuously exposed to 1.7 GHz LTE RF-EMF for 72 h at 1 and 2 SAR, cell proliferation was consistently decreased in all the human cells. The anti-proliferative effect was higher at 2 SAR than 1 SAR and was less severe in ASCs. The exposure to RF-EMF for 72 h at 1 and 2 SAR did not induce DNA double strand breaks or apoptotic cell death, but did trigger a slight delay in the G1 to S cell cycle transition. Cell senescence was also clearly observed in ASC and Huh7 cells exposed to RF-EMF at 2 SAR for 72 h. Intracellular ROS increased in these cells and the treatment with an ROS scavenger recapitulated the anti-proliferative effect of RF-EMF. These observations strongly suggest that 1.7 GHz LTE RF-EMF decrease proliferation and increase senescence by increasing intracellular ROS in human cells.

Adult stem cells are capable of self-renewal and differentiation into specific cell types in tissues and have high potential for stem cell therapy. Mesenchymal and hematopoietic stem cells are easily attainable from the human body and have become applicable tools for adult stem cell therapy. However, there are still technical barriers for the application of mesenchymal and hematopoietic stem cells for therapy, such as the small number of cell populations, high risk of contamination, and loss of their stemness properties in vitro. In our previous study, we showed that non-thermal atmospheric pressure plasma (NTAPP) promoted the proliferation of adipose tissue-derived stem cells (ASCs) by 1.6-fold on average, while maintaining their stemness. Here, we examined the feasibility of NTAPP as a tool to activate the proliferation of mesenchymal and hematopoietic stem cells in vitro without affecting their stem cell characteristics. NTAPP increased the proliferation of bone marrow-derived stem cells (BM-MSCs) and hematopoietic stem cells (HSCs) by 1.8- and 2-fold, respectively, when compared to that of untreated cells. As observed in ASCs, NTAPP exposure also activated the expression of stem cell-specific surface markers, CD44 and CD105, by 5-fold in BM-MSCs, when compared to that in unexposed control cells in a low glucose medium with a low concentration of basic fibroblast growth factor (b-FGF). In addition, NTAPP exposure highly augmented the mRNA expression of well-known pluripotent genes for stemness, such as Oct4, Sox2, and Nanog in ASCs and BM-MSCs when compared to that in unexposed control cells. When cell cycle progression was examined, the G1-S shift was accelerated, and expression of PCNA was increased in NTAPP-exposed ASCs when compared to that in untreated control cells, suggesting that NTAPP activated G1-S transition. Taken together, these results demonstrated that NTAPP activated the proliferation of various mesodermal-derived human adult stem cells by accelerating the G1-S transition while maintaining their pluripotency and stemness, strongly suggesting that NTAPP can be an efficient tool for expanding the population of various adult stem cells in vitro for medical applications.