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.
Highlights
The results show that process #3 is initiated by primary singlet oxygen from the gaseous phase of cold atmospheric plasma (CAP), independent of H2O2/NO2−-dependent primary 1O2 and independent of secondary 1O2, as it was not inhibited by AEBSF
The established scheme on the generation of primary and secondary 1O2 through, defined CAP- and plasma-activated medium (PAM)-derived compounds[59,60], the availability of scavengers and inhibitors that interfere with defined players in this signaling system (Fig. 1), and the possibility to dissect the system into defined steps or to reconstitute defined conditions[59,60], allowed us to elucidate the dynamics of CAP and PAM-mediated apoptosis induction in tumor cells
Treatment of cells with CAP in the presence of medium, or initial treatment of medium, followed by transfer of this plasma-activated medium (PAM) to cells, allowed to elucidate defined signaling processes, when this treatment was combined with the variation of incubation times, washing steps, as well as the addition or removal of inhibitors and dissecting/reconstituting the experimental system
Summary
Freund et al.[61] presented strong evidence that plasma-treated saline promotes an immunogenic phenotype of colon cancer cells, whereas Lin et al.[62] concluded that PAM is not sufficient to induce immunogenic cell death, but that short-lived species in CAP are required The discrepancy between these two studies might be explained by the low degree of induction of cell death by PAM in the study by Lin et al.[62]. Our preceding study has shown that the direct effect of short-lived singlet oxygen (1O2) from the gaseous phase of CAP on treated cells seemed to be neglectable compared to the effects that can be attributed to PAM-related compounds This finding can be explained by the preferential reaction (i.e. quenching) of 1O2 with medium components above or around the cells. This step is limited by the high reactivity of 1O2 and the resultant small free diffusion path length, due to 1O2 reaction with other competing substrates
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