Fig. 1.
Non-thermal plasma (NTP)-activated Ringer's lactate (PAL) reveals selective, ferroptosis-inducing activity on malignant mesothelioma (MM) cells in comparison to non-tumorous mesothelial cells. (A) Schematic illustration of experimental design. Briefly, a total of 10 mL of Ringer's lactate (RL) solution was placed in 60 mm-diameter culture dishes and exposed to NTP for 10 min, which was diluted to 14-fold with untreated Ringer's solution and used as PAL. The cells were treated with PAL for 15 min, which was further incubated in RPMI1640 medium (5% FBS). Control cells were treated with RL solution for 15 min and further incubated in RPMI1640 (5% FBS). (B) Differential effects of PAL between cancer and non-cancerous cells. Human non-tumorous mesothelial cells (LP9) and MM cells (H290, sarcomatoid subtype) were treated with PAL generated under different atmospheric composition for 15 min to evaluate cell proliferation after an incubation of 0–24 h/37 °C in RPMI (5% FBS). (C) Protective effects of Ferrostain-1 (Fer-1) only on cancer cells exposed to PAL. LP9 and MM cells (H290; 8A, epithelioid subtype) were treated with or without PAL (10% N2/10% O2/80% Ar) for 15 min to assess cell proliferation with or without Fer-1 (means ± SEM, N = 3; *P < 0.05, **P < 0.01, ***P < 0.001 vs RL unless indicated by bar). (D) PAL but not cisplatin induces ferroptosis in MM by dead cell analysis with SYTOX green and Hoechst 33342 nuclear staining under fluorescent microscopy. H290 cells were treated either with PAL (15 min), followed by an incubation of 6 h/37 °C in RPMI (5% FBS) with or without Fer-1, or with cisplatin in the presence or absence of Fer-1 for 6 h (bar = 50 μm). (E) FACS analysis of the experiments in D. Typical flow cytometry profiles are shown. (F) Transmission electron microscopy reveals (i) nuclear and (ii) mitochondrial alterations in H290 cells after PAL exposure, which are reversed by Fer-1 (bar = 5 μm at [i] and 500 nm at [ii]). Refer to text for details. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)