Antitumor effects of plasma-activated sodium acetate solution on gastric cancer cells

Abstract

Liquids irradiated with nonequilibrium atmospheric pressure plasma exert antitumor effects. Here, we produced plasma-activated acetated Ringer (PAA) and plasma-activated sodium acetate (PASA) solutions, each at 1%, 3%, and 5% mass concentrations. We evaluated the antitumor effects of PAA and PASA on gastric cancer (GC). Two GC cell lines (MKN1-Luc and MKN45-Luc) as well as normal human peritoneal mesothelial cells were subjected to cell viability assays using PAA, 1% PASA, 3% PASA, and 5% PASA. To elucidate the functional mechanisms, we examined morphological changes induced by 3% PASA following 10 min of irradiation. To further elucidate the underlying biological processes, we compared the expression of apoptosis-related proteins following the administration of 3% sodium acetate solution without plasma exposure and 3% PASA irradiated for 10 min. Additionally, MKN45-Luc cells were intraperitoneally injected into mice, followed by intraperitoneal administration of acetated Ringer's solution without plasma exposure (control-1 group), 3% sodium acetate solution without plasma exposure (control-2 group), and 3% PASA irradiated for 10 min (treatment group). Peritoneal dissemination was observed using in vivo bioluminescent imaging and laparotomy. PAA and PASA achieved an antitumor effect in a sodium acetate concentration-dependent manner. PAA and 3% PASA caused significantly less damage to normal peritoneal mesothelial cells compared to GC cells at 5 and 10 min of plasma exposure (p < 0.001). Blebs, indicative of apoptosis, were observed at 1.5 h after 3% PASA treatment in GC cells. 3% PASA treatment increased the expression of phosphorylated MKK3/MKK6 and phosphorylated p38 MAPK, suggesting that apoptosis may be mediated through the p38 MAPK pathway. The intraperitoneal administration of 3% PASA significantly reduced the number of peritoneal nodules, and no adverse events were detected. Here we show that PASA exerted an antitumor effect on GC, indicating that the intraperitoneal administration of 3% PASA may serve as a novel treatment for the peritoneal dissemination of GC.

Keywords: Gastric cancer; Nonequilibrium atmospheric pressure plasma; Plasma-activated sodium acetate.

Fig. 1

(a) The experimental system employed to produce plasma-activated liquids. L represents the distance between the plasma source and medium, and V represents the volume of the irradiated medium. In this experiment, L was fixed at 3 mm, and V at 6 ml. (b) Antitumor effects of PAL, PAA, 1% PASA, 3% PASA, and 5% PASA on GC cell lines. PASA had stronger antitumor effects at T = 0.5, 1, and 3 min compared with PAL. (c) Effects of PAL, PAA, 3% PASA, and 5% PASA on human peritoneal mesothelial cells. PAA and 3% PASA caused much less damage to normal peritoneal mesothelial cells compared with PAL. Error bars indicate standard deviation.

Fig. 2

(a) Apoptosis assay of normal peritoneal mesothelial cells and GC cells treated with 3% PASA. The percentages of apoptotic plus dead MKN1-Luc and MKN45-Luc cells increased within T = 3 min. (b) Morphological change induced by 3% PASA treatment. Morphological changes in MKN45-Luc cells by treatment with 3% sodium acetate solutions without plasma exposure (control group) or 3% PASA for T = 10 min (treatment group) were observed using time-lapse photography. In the treatment group, numerous blebs, indicative of apoptosis, were observed around the cells (arrow).

Fig. 3

(a) H₂O₂ and NO₂^- concentrations of PAL, PAA, 1% PASA, 3% PASA, and 5% PASA. H₂O₂ and NO₂^- concentrations of each plasma-activated solution increased approximately linearly with increasing plasma exposure time. (b) Na⁺, K⁺, glucose, and lactate concentrations of PAL, PAA, 1% PASA, 3% PASA, and 5% PASA. Glucose and lactate were not originally present in acetated Ringer’s and sodium acetate solutions without plasma exposure but were newly generated after plasma exposure. (c) Simple Western assay of MKN1-Luc and MKN-45-Luc cells treated with 3% sodium acetate solution without plasma exposure (control group) or 3% PASA at T = 10 min (treatment group). The expressions of phosphorylated MKK3/MKK6 and phosphorylated p38 MAPK were increased in the treatment group.

Fig. 4

Antitumor effect of 3% PASA on mouse xenograft models of peritoneal dissemination of GC. The intraperitoneal administration of 1.5 mL of acetated Ringer’s solution without plasma exposure (control-1 group), 3% sodium acetate solution without plasma exposure (control-2 group), or 3% PASA with a 10-min plasma exposure (treatment group) was performed on days 1–4 and 8–11. (a) Experimental protocol. (b) IVIS imaging data. (c) Laparotomy findings. The average number of peritoneal nodules in the treatment group was significantly smaller than that in the control-1 and control-2 groups. *P < 0.05. Error bars indicate standard deviation.