Dynamics of Singlet Oxygen-Triggered, RONS-Based Apoptosis Induction after Treatment of Tumor Cells with Cold Atmospheric Plasma or Plasma-Activated Medium

Abstract

Treatment of tumor cells with cold atmospheric plasma (CAP) or with plasma-activated medium (PAM) leads to a biochemical imprint on these cells. This imprint is mediated by primary singlet oxygen, which is mainly generated through the interaction between CAP-derived H2O2 and NO2−. This imprint is induced with a low efficiency as local inactivation of a few membrane-associated catalase molecules. As sustained generation of secondary singlet oxygen by the tumor cells is activated at the site of the imprint, a rapid bystander effect-like spreading of secondary singlet oxygen generation and catalase inactivation within the cell population is thus induced. This highly dynamic process is essentially driven by NOX1 and NOS of the tumor cells, and finally leads to intercellular RONS-driven apoptosis induction. This dynamic process can be studied by kinetic analysis, combined with the use of specific inhibitors at defined time intervals. Alternatively, it can be demonstrated and quantified by transfer experiments, where pretreated cells are mixed with untreated cells and bystander signaling is determined. These studies allow to conclude that the specific response of tumor cells to generate secondary singlet oxygen is the essential motor for their self-destruction, after a singlet oxygen-mediated triggering process by CAP or PAM.

Molecular Mechanisms of the Efficacy of Cold Atmospheric Pressure Plasma (CAP) in Cancer Treatment

“…The oxidative stress caused by plasma treatment, influences or damages these signaling molecules [102][103][104]. Alternatively, apoptosis may occur in neighboring cells due to the formation of secondary oxygen and the inactivation of the membrane-bound catalase [105,106]. It has also been shown that calcium ions can be transported from apoptotic to non-apoptotic neighbour cells via gap junctions, which also explains the widespread effect of plasma [107].…”

Modifying the Tumour Microenvironment: Challenges and Future Perspectives for Anticancer Plasma Treatments

“…The expression of soluble cues, such as chemokines, cytokines, and growth factors, are directly affected by the oxidative stress induced by plasma, as demonstrated before in cancer and immune cells [134,194,195]. In particular, tumour cells exposed to plasma or plasma-treated medium can induce a bystander effect that leads to cell death in the untreated neighbouring population, a process mediated by the generation of secondary 1 O 2 and inactivation of membrane-bound catalase, as discussed in Section 2 [60,97]. Previous studies have reported that gap junctions can propagate cell death signals by passing Ca 2+ ions from apoptotic to non-apoptotic neighbouring cancer cells [196], which could also explain the effect of plasma [197].…”

“…Depending on the plasma treatment conditions, 1 O 2 could either be generated directly and transferred from the gaseous phase to the liquid phase of the ECM or generated indirectly from H 2 O 2 and NO 2 -(which are transferred from the gaseous phase to the ECM). The latter scenario, where so-called "primary" 1 O 2 is generated in the ECM, has been elucidated to be the most likely [59,60]. Indeed, the formation of such primary 1 O 2 from a solution of H 2 O 2 and NO 2 has been experimentally verified [96,97].…”

“…The origin of the ROS triggering apoptosis in cancer cells after plasma treatment has been under debate: Although it would be logical to assume that the ROS were generated by plasma and added exogenously, it is true that some of these ROS have a very short life time and diffusion length due to their highly reactive nature and will not be able to reach the ECM, particularly not in the bulk of a cancer tumour. A paradigm first proposed in [58] and recently experimentally verified [59,60], states that the ROS acting in the ECM of cancer cells after plasma treatment instead are generated by the cells themselves and are part of a system of signalling pathways used by cancer cells to promote proliferation. In this scenario, the effect that plasma exerts on cancer tumour is constituted by the transmission and/or creation of one species, 1 O 2 , to the ECM.…”

“…The generation of secondary 1 O 2 occurs in an exponential manner and creates an amplified apoptosis signal reaching the bulk of the tumour. The whole set of events-from the generation of primary 1 O 2 , catalase-inactivation, and subsequently, tumour-generated secondary 1 O 2 (followed by inactivation of more catalase), to apoptosis-induction-has been experimentally investigated and verified both in the case of tumour cells in a solution of H 2 O 2 and NO 2 - [96,97] and for plasma-treated tumour cells [59,60]. The selectivity of the treatment, given that the concentration of H 2 O 2 is below a certain limit, has been confirmed by control experiments with non-malignant cells [59].…”