Figure 8

Kinetic and mechanistic aspects of different modes of CAP treatment of tumor cells. I. (A) MKN-45 cells remained untreated and were incubated for 4 h. (B) Medium and cells were treated with CAP for 1 min, followed by 25 min of incubation, a washing step and further incubation in fresh medium up to 4 h; (C) The NOX1 inhibitor AEBSF (100 µM) was present during the preatreatment of cells + medium with CAP for 1 min, followed by 25 min incubation, a washing step and further incubation in fresh medium. (D) Medium was treated with CAP for 1 min and then tumor cells were added to reach a final concentration of PAM of 80%. The assays were further incubated up to 4 h. (E) Medium was treated CAP for 1 min and then tumor cells were added. The assays were further incubated for 25 min, washed, and further incubated in fresh medium up to 4 h; (F) Medium plus cells were treated with CAP for 1 min and then washed immediately, resuspended in fresh medium and further cultivated. (G) The assay was performed as described undeer F, with the modification that AEBSF was present during CAP treatment. II., III.: The conditions and assays are identical to those described under I, but the time of assessment of apoptosis was at 6 h or 7.5 h after CAP treatment, respectively. The results show that CAP treatment of tumor cells in medium, followed by 25 min incubation in the same medium causes apoptosis induction to the same degree as treatment of medium with CAP, followed by incubation of cells in this plasma-activated medium (PAM). This points to the dominant action of long-lived species for apoptosis induction. The presence of AEBSF during CAP treatment and 25 min incubation causes a kinetic delay, which is explained by initial prevention of secondary singlet oxygen (¹O₂) generation, followed by resumption of secondary ¹O₂ generation after the washing step and incubation in fresh medium. Separation of the CAP-treated cells from their medium immediately after 1 min CAP treatment results in a very long delay in apoptosis induction, as ¹O₂ from the gaseous phase of CAP is the only initial trigger under the conditions in assays F and G.