To date, the significant anti-cancer capacity of cold atmospheric plasma (CAP) on dozens of cancer cell lines has been demonstrated in vitro and in mice models. Conventionally, CAP was directly applied to irradiate cancer cells or tumor tissue. Over past three years, the CAP irradiated media was also found to kill cancer cells as effectively as the direct CAP treatment. As a novel strategy, using the CAP stimulated (CAPs) media has become a promising anti-cancer tool. In this study, we demonstrated several principles to optimize the anti-cancer capacity of the CAPs media on glioblastoma cells and breast cancer cells. Specifically, using larger wells on a multi-well plate, smaller gaps between the plasma source and the media, and smaller media volume enabled us to obtain a stronger anti-cancer CAPs media composition without increasing the treatment time. Furthermore, cysteine was the main target of effective reactive species in the CAPs media. Glioblastoma cells were more resistant to the CAPs media than breast cancer cells. Glioblastoma cells consumed the effective reactive species faster than breast cancer cells did. In contrast to nitric oxide, hydrogen peroxide was more likely to be the effective reactive species.

Selectively treating tumor cells is the ongoing challenge of modern cancer therapy. Recently, cold atmospheric plasma (CAP), a near room-temperature ionized gas, has been demonstrated to exhibit selective anticancer behavior. However, the mechanism governing such selectivity is still largely unknown. In this review, the authors first summarize the progress that has been made applying CAP as a selective tool for cancer treatment. Then, the key role of aquaporins in the H2O2 transmembrane diffusion is discussed. Finally, a novel model, based on the expression of aquaporins, is proposed to explain why cancer cells respond to CAP treatment with a greater rise in reactive oxygen species than homologous normal cells. Cancer cells tend to express more aquaporins on their cytoplasmic membranes, which may cause the H2O2 uptake speed in cancer cells to be faster than in normal cells. As a result, CAP treatment kills cancer cells more easily than normal cells. Our preliminary observations indicated that glioblastoma cells consumed H2O2 much faster than did astrocytes in either the CAP-treated or H2O2-rich media, which supported the selective model based on aquaporins.

Over past several years, the cold plasma-stimulated medium (PSM) has shown its remarkable anti-cancer capacity in par with the direct cold plasma irradiation on cancer cells or tumor tissues. Independent of the cold plasma device, PSM has noticeable advantage of being a flexible platform in cancer treatment. Currently, the largest disadvantage of PSM is its degradation during the storage over a wide temperature range. So far, to stabilize PSM, it must be remained frozen at −80 °C. In this study, we first reveal that the degradation of PSM is mainly due to the reaction between the reactive species and specific amino acids; mainly cysteine and methionine in medium. Based on this finding, both H2O2 in PSM and the anti-cancer capacity of PSM can be significantly stabilized during the storage at 8 °C and −25 °C for at least 3 days by using phosphate-buffered saline (PBS) and cysteine/methionine-free Dulbecco’s Modified Eagle Medium (DMEM). In addition, we demonstrate that adding a tyrosine derivative, 3-Nitro-L-tyrosine, into DMEM can mitigate the degradation of PSM at 8 °C during 3 days of storage. This study provides a solid foundation for the future anti-cancer application of PSM.

Cold atmospheric plasma (CAP), a novel promising anti-cancer modality, has shown its selective anti-cancer capacity on dozens of cancer cell lines in vitro and on subcutaneous xenograft tumors in mice. Over the past five years, the CAP-stimulated solutions (PSS) have also shown their selective anti-cancer effect over different cancers in vitro and in vivo. The solutions used to make PSS include several bio-adaptable solutions, mainly cell culture medium and simple buffered solutions. Both the CAP-stimulated medium (PSM) and the CAP-stimulated buffered solution (PSB) are able to significantly kill cancer cells in vitro. In this study, we systematically compared the anti-cancer effect of PSM and PSB over pancreatic adenocarcinoma cells and glioblastoma cells. We demonstrated that pancreatic cancer cells and glioblastoma cells were specifically vulnerable to PSM and PSB, respectively. The specific response such as the rise of intracellular reactive oxygen species of two cancer cell lines to the H2O2-containing environments might result in the specific vulnerabilities to PSM and PSB. In addition, we demonstrated a basic guideline that the toxicity of PSS on cancer cells could be significantly modulated through controlling the dilutability of solution.

The cold atmospheric plasma (CAP) is a promising novel anti-cancer method. Our previous study showed that the cold plasma-stimulated medium (PSM) exerted remarkable anti-cancer effect as effectively as the direct CAP treatment did. H 2 O 2 has been identified as a key anticancer substance in PSM. However, the mechanisms underlying intracellular H 2 O 2 regulation by cancer cells is largely unknown. Aquaporins (AQPs) are the confirmed membrane channels of H 2 O 2 . In this study, we first demonstrated that the anti-glioblastoma capacity of PSM could be inhibited by silencing the expression of AQP8 in glioblastoma cells (U87MG) or using the aquaporins-blocker silver atoms. This discovery illustrates the key intermediate role of AQPs in the toxicity of PSM on cancer cells. Because the expression of AQPs varies significantly among different cancer cell lines, this study may facilitate the understanding on the diverse responses of cancer cells to PSM or the direct CAP treatment.