Non-Thermal Atmospheric Pressure Bio-Compatible Plasma Stimulates Apoptosis via p38/MAPK Mechanism in U87 Malignant Glioblastoma

Abstract

Nonthermal plasma is a promising novel therapy for the alteration of biological and clinical functions of cells and tissues, including apoptosis and inhibition of tumor progression. This therapy generates reactive oxygen and nitrogen species (RONS), which play a major role in anticancer effects. Previous research has verified that plasma jets can selectively induce apoptosis in various cancer cells, suggesting that it could be a potentially effective novel therapy in combination with or as an alternative to conventional therapeutic methods. In this study, we determined the effects of nonthermal air soft plasma jets on a U87 MG brain cancer cell line, including the dose- and time-dependent effects and the physicochemical and biological correlation between the RONS cascade and p38/mitogen-activated protein kinase (MAPK) signaling pathway, which contribute to apoptosis. The results indicated that soft plasma jets efficiently inhibit cell proliferation and induce apoptosis in U87 MG cells but have minimal effects on astrocytes. These findings revealed that soft plasma jets produce a potent cytotoxic effect via the initiation of cell cycle arrest and apoptosis. The production of reactive oxygen species (ROS) in cells was tested, and an intracellular ROS scavenger, N-acetyl cysteine (NAC), was examined. Our results suggested that soft plasma jets could potentially be used as an effective approach for anticancer therapy.

Keywords: human glioblastoma; nonthermal biocompatible plasma; p38/MAPK pathway; reactive oxygen and nitrogen species; soft jet plasma.

Figure 1

(A) Schematic overview of the experimental setup; (B) measurement of optical emission spectra (OES) of the soft plasma jet device with air gas flow.

Figure 2

(A) Morphological changes in cell lines of astrocytes and U87 MG cells treated with plasma for 30, 60, and 180 s and viewed under a light microscope. Arrows indicate cell shrinkage and nuclear condensation due to apoptosis and the presence of apoptotic bodies. (B) SEM morphology of astrocytes and U87 MG cells. Effects of a soft plasma jet on cell viability of astrocytes (C) and U87 MG cells (D). (E) Confocal microcopy images of intracellular reactive oxygen species (ROS) and intracellular reactive nitrogen species (RNS) of U87 MG cells. (F) Relative amounts of ROS and RNS levels following soft plasma jet treatment of U87 MG cells. Scale bar = 100 μm. All values are presented as means ± SD of three independent experiments. ** p < 0.01, and *** p < 0.001.

Figure 3

Apoptosis in astrocytes and U87 MG cells after plasma treatment: (A) representative flow cytometry (fluorescence-activated cell sorting (FACS)) dot plots of astrocytes and U87 MG cells prepared using the Annexin V-FITC Apoptosis Detection Kit; (B) summaries of frequencies of early and late apoptosis events for astrocytes and U87 MG cells; (C) effects of a soft plasma jet on the cell cycle distribution of U87 MG human brain cancer cells; (D) the percentage of cells in different cell cycle phases. The cells were treated for 30, 60, and 180 s and analyzed by flow cytometry. All values are presented as means ± SD of three independent experiments. ** p < 0.01, and *** p < 0.001.

Figure 4

(A) Western blot analysis of protein expression in U87 MG cells; (B) relative band intensity as a function of treatment time.

Figure 5

In vivo targeting of glioblastoma tumor with nonthermal atmospheric biocompatible plasma (NBP): (A) representative bioluminescence imaging; (B) region of interest (ROI) levels for changes in the tumor; (C) tumor size of the sectioned mouse brain by bioluminescence imaging and tumor volume; (D,E) expression of p-p38, cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), and survivin determined via an immunofluorescence assay in mouse tumor tissues. All values are presented as means ± SD of three independent experiments. ** p < 0.01, and *** p < 0.001.