Anticancer Effects of Plasma-Activated Medium Produced by a Microwave-Excited Atmospheric Pressure Argon Plasma Jet

Abstract

Cold atmospheric plasma (CAP) has been reported to have strong anticancer effects in vitro and in vivo. CAP has been known to induce apoptosis in most cancer cells by treatment to cells using direct and indirect treatment methods. There are many reports of apoptosis pathways induced by CAP, but for indirect treatment, there is still a lack of fundamental research on how CAP can cause apoptosis in cancer cells. In this study, we applied an indirect treatment method to determine how CAP can induce cancer cell death. First, plasma-activated medium (PAM) was produced by a 2.45 GHz microwave-excited atmospheric pressure plasma jet (ME-APPJ). Next, the amounts of various reactive species in the PAM were estimated using colorimetric methods. The concentration of NO2– and H2O2 in PAM cultured with cancer cells was measured, and intracellular reactive oxidative stress (ROS) changes were observed using flow cytometry. When PAM was incubated with A549 lung cancer cells, there was little change in NO2– concentration, but the concentration of H2O2 gradually decreased after 30 min. While the intracellular ROS of A549 cells was rapidly increased at 2 hours, there was no significant change in that of PAM-treated normal cells. Furthermore, PAM had a significant cytotoxic effect on A549 cells but had little effect on normal cell viability. In addition, using flow cytometry, we confirmed that apoptosis of A549 cells occurred following flow cytometry and western blot analysis. These results suggest that among various reactive species produced by PAM, hydrogen peroxide plays a key role in inducing cancer cell apoptosis.

Role of Short- and Long-Lived Reactive Species on the Selectivity and Anti-Cancer Action of Plasma Treatment In Vitro

“…Most of the reactive species found in the liquid phase are either primarily generated in the gas phase and at the gas-liquid interface or are the end-products of the transformation of primary ROS/RNS in liquid [30,31,38,39]. While H 2 O 2 is a central player in the anti-cancer capacity of CAP treatment [40][41][42][43][44][45][46][47][48][49], we, and others, have shown that H 2 O 2 and NO 2 − act synergistically to induce cell death after plasma treatment [46,50,51].…”

“…While several groups have shown that, in vitro, both treatments were equivalent in inducing cancer cell death, in altering cell surface adhesion molecules or in inactivating enzymatic functions [46,[56][57][58], others have reported that direct treatment is more effective than indirect treatment at killing tumor cells [59][60][61][62][63]. Moreover, there is still a debate regarding the selectivity of cold plasma at inducing cell death preferentially in tumor cells over healthy cells [46,49,[64][65][66][67][68][69][70][71]. These discrepancies between reports can be attributed to the fact that the anti-cancer capacity of plasma-activated liquid depends on several factors such as the size of the wells in which cells are seeded, the volume of the treated liquid, the liquid composition (e.g., PBS versus culture medium), the gap between the plasma source and the liquid, the gas flow rate, the gas admixture, and the plasma device itself [48,[71][72][73].…”

The Effect of Plasma Activated Medium and PBS on Human Melanoma Cells Compared With Other Cancer and Normal Cells

“…Different types of human fibroblasts were used in studies and it was shown that the treatment with PAM was better tolerated than the direct plasma treatment [38]. PAM activated by He plasma jet decreased the Nuff fibroblasts viability by 20% [39] and 3T3 fibroblasts, if PAM was activated for more than 30 seconds [40]. PAM activated by Ar plasma reduced the viability of WI-38 by 40% [26], however, it had no effect on mammary epithelium cells [41] or human astrocytes [24].…”

Intracellular Responses Triggered by Cold Atmospheric Plasma and Plasma-Activated Media in Cancer Cells

“…During cell/tissue treatment with CAP or PAM, cellular and organelles membranes represent the natural interphase, which first comes in contact with the above mentioned RONS produced in PAM or within the cells. The membranes allow for the translation of RONS chemical reactivity into distinct biological responses [ 38 ]. Tumour cells are protected against intercellular apoptosis by inducing signalling with increased expression of membrane-associated enzymes catalase and superoxide dismutases [ 39 ].…”

“…The exposure to CAP or PAM initially inactivates only a small percentage of protective membrane-associated catalase molecules in the tumour cells [ 28 ]. Then the tumour cells efficiently propagate their cell death through their own CAP-induced RONS signalling [ 38 , 44 ].…”