Anticancer Effects of Plasma-Treated Water Solutions from Clinically Approved Infusion Liquids Supplemented with Organic Molecules

Abstract

Water solutions treated by cold atmospheric plasmas (CAPs) currently stand out in the field of cancer treatment as sources of exogenous blends of reactive oxygen and nitrogen species (RONS). It is well known that the balance of RONS inside both eukaryotic and prokaryotic cells is directly involved in physiological as well as pathological pathways. Also, organic molecules including phenols could exert promising anticancer effects, mostly attributed to their pro-oxidant ability in vitro and in vivo to generate RONS like O₂^-, H₂O₂, and a mixture of potentially cytotoxic compounds. By our vision of combining the efficacy of plasma-produced RONS and the use of organic molecules, we could synergistically attack cancer cells; yet, so far, this combination, to the best of our knowledge, has been completely unexplored. In this study, l-tyrosine, an amino acid with a phenolic side chain, is added to a physiological solution, often used in clinical practice (SIII) to be exposed to plasma. The efficacy of the gas plasma-oxidized SIII solution, containing tyrosine, was evaluated on four cancer cell lines selected from among tumors with poor prognosis (SHSY-5Y, MCF-7, HT-29, and SW-480). The aim was to induce tumor toxicity and trigger apoptosis pathways. The results clearly indicate that the plasma-treated water solution (PTWS) reduced cell viability and oxygen uptake due to an increase in intracellular ROS levels and activation of apoptosis pathways in all investigated cancer cells, which may be related to the activation of the mitochondrial-mediated and p-JNK/caspase-3 signaling pathways. This research offers improved knowledge about the physiological mechanisms underlying cancer treatment and a valid method to set up a prompt, adequate, and effective cancer treatment in the clinic.

Figure 1

Experimental apparatus and scheme of the research. (A) Schematic overview of the DBD plasma source; (B) image of the discharge unit with plasma ignited; (C) scheme of the production of PTWS and their chemical/biological characterization. A confocal microscope image of cancer cells and an AFM image of the FC-PLL membrane used as substrate for cell seeding are shown on the right side of the scheme.

Figure 2

Production of RONS. H₂O₂ and NO₂^– concentrations generated in SIII without (A–C) and with the addition of tyrosine (300 mg/L) (B–D), after igniting air- or O₂-fed DBDs for different treatment times. DBDs were ignited at 13.8 kV, 6 kHz, 25% DC, 0.5 slm flow rate in 2 mL of liquids 3 mm far from the ground electrode.

Figure 3

Analysis of RONS in SIII-tyr solutions. Concentration of (A) H₂O₂ and (B) NO₂ generated in SIII at increasing concentrations (0–300 mg/L) of added tyrosine, after 3 min of air- or O₂-fed DBD (13.8 kV, 6 kHz, 25% DC, 0.5 slm feed flow rate, 2 mL liquids 3 mm far from the ground electrode).

Figure 4

LC-MS of SIII and SIII-tyr solutions. Positive (A, C, E, G) and negative (B, D, F, H) mode spectra acquired for (A, B) untreated SIII; (C, D) untreated SIII-tyr; (E, F) SIII-tyr treated with 45 s of O₂ plasma; and (G, H) SIII-tyr treated with 3 min air plasma (13.8 kV, 6 kHz, 25% DC, 0.5 slm feed flow rate, 2 mL liquids 3 mm far from the ground electrode).

Figure 5

Aging of PTWS and mock solutions in different storage environments. Concentration of H₂O₂ at (A) 25 °C and (B) 4 °C and (C) concentration of nitrites at different storage temperatures. PTWS obtained with 3 min air- or O₂-DBD (13.8 kV, 6 kHz, 25% DC, 0.5 slm flow rate on 2 mL of liquids 3 mm far from the ground electrode) were compared with a mock solution obtained by dissolving H₂O₂ in SIII + tyr solution with a final concentration of 80 μM.

Figure 6

Antiproliferative effect of PTWS in cancer cells. Cell viability after 24, 48, and 72 h of 2-h incubation in SIII-tyr, air-DBD, and O₂-DBD. Data statistically significant according to ANOVA followed by Bonferroni t-test (p < 0.05). * vs air-DBD and O₂-DBD at the same culture time; ^§ vs 24 h for the same treatment; ^θ vs 48 and 72 h for the same treatment; ^⧧ vs air-DBD at the same culture time; ^ηvs 72 h for the same treatment.

Figure 7

Pro-oxidant effect of PTWS. (A–C) Confocal laser micrographs of the DCF fluorescence signal in MCF-7 (A), HT-29 (B), and SW-480 (C) cancer cells 72 h after 2-h incubation in different solutions with respect to cells grown on untreated medium used as control (CNTR). Scale bar: 20 μm. (D–F) Quantitative analysis of the DCF fluorescence intensity produced in MCF-7 (D), HT-29 (E), and SW-480 (F) tumor cells 24, 48, and 72 h after 2-h incubation in different solutions with respect to cells grown on untreated medium used as control (CNTR). Data statistically significant according to one-way ANOVA followed by Bonferroni t-test (p < 0.05). *vs CNTR and SIII-tyr at the same culture time; ^⧧vs all treatments at the same culture time.

Figure 8

Study of mitochondrial membrane potential and activation of apoptotic markers. Pro-mitochondrial membrane potential and apoptotic effect of PTWS in MCF-7 (A, D, G), HT-29 (B, E, H), and SW-480 (C, F, I) cancer cells, after 24, 48, and 72 h of 2-h treatment with SIII-tyr, air-DBD, and O₂-DBD. (A–C) Quantitative analysis of the fluorescence of JC-1 red and green intensity ratio. (D–I) Quantitative analysis of the activation of apoptotic markers caspase-3 (D–F) and p-JNK (G–I). The percentage of apoptotic cells was calculated by the ratio of apoptotic cells (active caspase-3-positive and p-JNK-positive) over total nuclei (DAPI-stained nuclei). The analyses on cells grown on untreated medium were used as control (CNTR). Data statistically significant according to ANOVA followed by a Bonferroni post-test (p < 0.05). *vs air-DBD and O₂-DBD for each culture time; ^§vs CNTR and SIII-tyr for each culture time; ^†vs all treatments at 72 h; ^#vs all treatments for each culture time; ^⧧vs all treatments at 24 h.

Figure 9

Metabolic activity. O₂ consumption in tumor cells 24, 48, and 72 h after 2-h exposure in SIII-tyr, air-DBD, and O₂-DBD. Data statistically significant according to ANOVA followed by Bonferroni t-test (p < 0.05). *vs air-DBD and O₂-DBD for each culture time; ^§vs O₂-DBD for each culture time; ^⧧vs O₂-DBD at 72 h; ^θvs air-DBD and O₂-DBD at 48 and 72 h; ^ωvs and O₂-DBD at 48 and 72 h.