Cold atmospheric plasma jet-generated RONS and their selective effects on normal and carcinoma cells

Abstract

Cold atmospheric helium plasma jets were fabricated and utilized for plasma-cell interactions. The effect of operating parameters and jet design on the generation of specific reactive oxygen and nitrogen species (RONS) within cells and cellular response were investigated. It was found that plasma treatment induced the overproduction of RONS in various cancer cell lines selectively. The plasma under a relatively low applied voltage induced the detachment of cells, a reduction in cell viability, and apoptosis, while the plasma under higher applied voltage led to cellular necrosis in our case. To determine whether plasma-induced reactive oxygen species (ROS) generation occurs through interfering with mitochondria-related cellular response, we examined the plasma effects on ROS generation in both parental A549 cells and A549 ρ(0) cells. It was observed that cancer cells were more susceptible to plasma-induced RONS (especially nitric oxide (NO) and nitrogen dioxide (NO2(-)) radicals) than normal cells, and consequently, plasma induced apoptotic cell responses mainly in cancer cells.

Figure 1. Plasma jet devices and electrical and optical characteristics.

(A) Schematic of experimental setup and structure of two different jet devices (Jet-Type 1 and 2). (B) Waveforms of discharge currents for Jet-Type 1 (upper row of figure) and Jet-Type 2 (bottom row of figure). (C) photograph and optical emission spectrum of plasma plume (upper row of figure: Jet-Type 1; bottom row of figure: Jet-Type 2) from 200–900 nm observed in plasma at applied voltage 1.8 kV_pp, repetition frequency 35 kHz, and duty ratio 8%.

Figure 2. Morphological change and reduction in viability of PC3 cancer cells (using Jet-Type 1).

(A) Bright field images of cells (left: gas-treated control; right: detachment from surface after plasma treatment (0.8 kV_rms) for 20 s). (B) Measurement of cell viability by MTS assay at 24 h after gas (10 s) and plasma treatment (0.8 kV_rms) for 5 and 10 s on five points (Each point represents mean ± SD of three replicates; **p < 0.01). Scale bar = 100 μm.

Figure 3. Intracellular ROS generation in cancer (A549) and normal (HCAEC) cells (using Jet-Type 1).

Fluorescence images and intensity graphs of intracellular ROS production after plasma treatment for 10 s (0.8 kV_rms) using DCF-DA assay and bright field images: (A) A549 cells (upper row: gas-treated control; bottom row: plasma treatment). (B) HCAEC cells (upper row: gas-treated control; bottom row: plasma treatment). Scale bar = 100 μm. (Each point represents mean ± SD of three replicates; ***p < 0.001).

Figure 4. Comparison between parental A549 and A549-ρ⁰ cells in plasma-induced ROS production (using Jet-Type 1).

Fluorescence images of intracellular ROS production after plasma treatment for 10 s (0.8 kV_rms) by using DCF-DA assay and bright field images: (A) A549 cells (upper row: gas-treated control; bottom row: plasma treatment) and (B) A549-ρ⁰ cells (upper row: gas-treated control; bottom row: plasma treatment). Scale bar = 100 μm. (C) Quantification by measuring fluorescence pixel intensity with MetaMorph software (Each point represents mean ± SD of three replicates; **p < 0.01). (D) Immunoblot of cytochrome c oxidase II (COX II) showing that this protein, which is coded by mtDNA, is present in A549 cells but not in A549-ρ⁰ cells. Blot was probed with anti-actin to ensure equal protein loading. (E) Fluorescence (left) and merged images with bright field image (right) of the mitochondria-staining in A549 cells (upper row) and in A549-ρ⁰ cells (bottom row, morphological change: white arrows).

Figure 5. Induction of necrotic and apoptotic cell death in A549 cancer cells (using Jet-Type 1).

(A) Merged image of fluorescence using DCF-DA with bright field images and intensity graphs after plasma treatment (1.2 kV_rms) for 10 s (upper row, left: gas-treated control, right: plasma-treatment) and cytotoxicity (% LDH) rate as a function of applied voltage (bottom row). Scale bar = 100 μm. (B) Measurement of cell viability by MTS assay at 24 and 48 h after gas and plasma treatment (0.9 and 1 kV_rms) for 10 s on five points. (C) Immunoblot of PARP at 24 h after gas and plasma treatment (1 kV_rms) for 10 s on nine points per 35-mm dish. Blot was probed with anti-actin to ensure equal protein loading. (D) The rate of cell viability by using Jet-Type 1 and 2 under the same parameter with a pulsed high-voltage supply as a function of applied voltage. (Each point represents mean ± SD of three replicates; *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 6. Intracellular RONS generation by plasma under various operating parameters in lung cancer (A549) and melanoma (SK-MEL2) cells (using Jet-Type 2).

Fluorescence images and intensity graphs of intracellular ROS production and bright field images: (A) DCF-DA assay after plasma treatment (1.7 kV_pp) (upper row: gas-treated control; bottom row: plasma treatment). (B) APF assay as a function of applied voltage (upper row: 1.6 kV_pp; bottom row: 1.9 kV_pp). (C) DAF-2DA assay after plasma treatment (1.7 kV_pp) as a function of decreasing distance from nozzle to cells (upper row: 17 mm; bottom row: 10 mm). Scale bar = 100 μm. (D) Measurement of nitrite concentration as a function of applied voltage (for 3 min with 3-mm-thick layer of HBSS; 1.6, 1.7, and 1.8 kV_pp) in A549 and SK-MEL2 cells by Griess assay (upper row) and fluorescence images and intensity graphs of intracellular NO production as a function of applied voltage (1.6 and 1.8 kV_pp) by DAF-2DA assay (middle row: A549; bottom row: SK-MEL2). (Each point represents mean ± SD of three replicates; *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 7. Induction of cellular apoptotic-like change (using Jet-Type 2).

(A) TUNEL-positive (green) apoptotic cells (red circles) and merged images (upper row: gas-treated control; bottom row: plasma treatment) by TUNEL assay at 48 h after plasma treatment (1.9 kV_pp) for 5 min on a dish (with 3-mm-thick layer of serum-free media). Cells were harvested by trypsinization 48 h after exposure to plasma and then fixed with 1% paraformaldehyde for 20 min in PBS. (B) The images with TUNEL-positive (green) apoptotic cells and cell nuclei (red) by PI staining (upper row: gas-treated control, bottom row: plasma-treatment). Cells stained weakly and irregularly by PI (red circles), indicating that these nuclei were fragmented after plasma exposure. Scale bar = 100 μm.

Figure 8. Level of intracellular OH and NO production in cancer and normal cells (using Jet-Type 2).

Fluorescence images and intensity graphs of intracellular ROS production after plasma treatment for 10 s (1.7 kV_pp) and bright field images: (A) APF assay (upper row: normal BEAS-2B; bottom row: cancer A549). (B) DAF-2DA (upper row: normal HS27; middle row: normal BEAS-2B; bottom row: cancer A549). Scale bar = 100 μm. (Each point represents mean ± SD of three replicates; ***p < 0.001).

Figure 9. Measurement of nitrite concentration and caspase 3/7 activation in cancer and normal cells (using Jet-Type 2).

(A) Measurement of nitrite concentration as a function of applied voltage (1.6, 1.7, and 1.8 kV_pp) in cancer A549 and normal BEAS-2B cells by Griess assay at 12 h after treatment for 3 min with 3-mm-thick layer of HBSS (each point represents mean ± SD of three replicates). Fluorescence images and intensity graphs of caspase 3/7 activation and bright field images in (B) BEAS-2B (upper row: gas-treated control; bottom row: plasma treatment) and (C) A549 cells (upper row: gas-treated control; bottom row: plasma treatment). Cells were loaded with CellEvent Caspase 3/7 Green Detection Reagent at 16 h after plasma treatment (1.8 kV_pp) for 10 s. Positive apoptotic cells containing active caspases 3 and 7 appear in green. Scale bar = 100 μm. (Each point represents mean ± SD of three replicates; *p < 0.05, **p < 0.01).