The Strong Cell-based Hydrogen Peroxide Generation Triggered by Cold Atmospheric Plasma

Abstract

Hydrogen peroxide (H₂O₂) is an important signaling molecule in cancer cells. However, the significant secretion of H₂O₂ by cancer cells have been rarely observed. Cold atmospheric plasma (CAP) is a near room temperature ionized gas composed of neutral particles, charged particles, reactive species, and electrons. Here, we first demonstrated that breast cancer cells and pancreatic adenocarcinoma cells generated micromolar level H₂O₂ during just 1 min of direct CAP treatment on these cells. The cell-based H₂O₂ generation is affected by the medium volume, the cell confluence, as well as the discharge voltage. The application of cold atmospheric plasma (CAP) in cancer treatment has been intensively investigated over the past decade. Several cellular responses to CAP treatment have been observed including the consumption of the CAP-originated reactive species, the rise of intracellular reactive oxygen species, the damage on DNA and mitochondria, as well as the activation of apoptotic events. This is a new previously unknown cellular response to CAP, which provides a new prospective to understand the interaction between CAP and cells in vitro and in vivo. The short-lived reactive species in CAP may activate cells in vivo to generate long-lived reactive species such as H₂O₂, which may trigger immune attack on tumorous tissues via the H₂O₂-mediated lymphocyte activation.

Figure 1

Only the direct CAP treatment can trigger the H₂O₂ production of specific cancer cell lines in vitro. (a) The H₂O₂ concentration in DMEM after the CAP treatment on just DMEM (control), on pancreatic adenocarcinoma cells (PA-TU-8988T) immerged in DMEM, on breast adenocarcinoma cells (MDA-MB-231) immerged in DMEM, as well as on glioblastoma cells (U87MG) immerged in DMEM. (b) The cells-based H₂O₂ concentration. The data is calculated based on the following formula. Cells-based H₂O₂ concentration = H₂O₂ concentration in the DMEM which has been used to immerse cancer cells during the CAP treatment – H₂O₂ concentration in the CAP-treated DMEM (control). The H₂O₂ concentration corresponds to the mean of each bar shown in 1a. (c) The indirect CAP treatment will not stimulate cancer cells to generate H₂O₂. All H₂O₂ concentration was measured immediately (about 1 min) after the treatment. Results are presented as the mean ± s.d. of three independently repeated experiments. Student’s t-test was performed between the data based on cells immersed in specific volume of DMEM and the data just based on the DMEM with the same volume. The significance is indicated as *p < 0.05.

Figure 2

H₂O₂ evolution in the DMEM surrounding cancer cells after the direct CAP treatment. (a) Cancer cells immersed in 20 μL of DMEM. (b) Cancer cells immersed in 100 μL of DMEM. CAP was used to treat DMEM (control) or cancer cells (MDA-MB-231 cells, PA-TU-8988T cells, and U87MG cells) immersed in DMEM. In each experiment, the 1 min of CAP treatment was performed on the cells immersed in 20 μL or 100 μL of DMEM. The H₂O₂ concentration was measured very minute after the CAP treatment until the tenth minute. Cancer cells in 96-wells plate were cultured overnight before the CAP treatment. The cell confluence was 3 × 10⁴ cells/mL. Results are presented as the mean ± s.d. of three independently repeated experiments.

Figure 3

The cell confluence and the output voltage affect the cell-based H₂O₂ generation. (a) A relative small confluence can achieve the maximum cell-based H₂O₂ generation. (b) High output AV voltage significantly weakens the cell-based H₂O₂ generation. In each experiment, a 1 min of CAP treatment was used to treat DMEM (control) or cancer cells (MDA-MB-231 cells, PA-TU-8988T cells, and U87MG cells) immersed in 100 μL of DMEM. The H₂O₂ concentration was measured immediately (about 1 min) after the treatment. Cancer cells were cultured overnight before the treatment. The cell confluence for (b) was 3 × 10⁴ cells/mL. Results are presented as the mean ± s.d. of three independently repeated experiments. Student’s t-test was performed and the significance is indicated as *p < 0.05.

Figure 4

Under the same experimental conditions, the anti-cancer capacity of the direct CAP treatment is significantly stronger than the indirect CAP treatment. For the indirect CAP treatment, the CAP-stimulated DMEM was used to affect the growth of cancer cells. Comparison between the direct CAP treatment and the indirect treatment is performed on (a) MDA-MB-231 cells and (b) PA-TU-8988T cells. Pre-treating cancer cells with 6 mM NAC an intracellular scavenger for 3 hr or immediately (about 1 min) renewing the DMEM after the direct CAP treatment can effectively weakens the anti-cancer capacity of the direct CAP treatment on (c) MDA-MB-231 cells and (d) PA-TU-8988T cells. Results are presented as the mean ± s.d. of three independently repeated experiments. Student’s t-test was performed between the results from the direct CAP treatment and the results from the indirect CAP treatment. The significance is indicated as *p < 0.05.