Cold atmospheric plasma induces apoptosis in human colon and lung cancer cells through modulating mitochondrial pathway

Abstract

Cold atmospheric plasma (CAP) is an emerging and promising oncotherapy with considerable potential and advantages that traditional treatment modalities lack. The objective of this study was to investigate the effect and mechanism of plasma-inhibited proliferation and plasma-induced apoptosis on human lung cancer and colon cancer cells in vitro and in vivo. Piezobrush^® PZ2, a handheld CAP unit based on the piezoelectric direct discharge technology, was used to generate and deliver non-thermal plasma. Firstly, CAP_PZ2 treatment inhibited the proliferation of HT29 colorectal cancer cells and A549 lung cancer cells using CCK8 assay, caused morphological changes at the cellular and subcellular levels using transmission electron microscopy, and suppressed both types of tumor cell migration and invasion using the Transwell migration and Matrigel invasion assay. Secondly, we confirmed plasma-induced apoptosis in the HT29 and A549 cells using the AO/EB staining coupled with flow cytometry, and verified the production of apoptosis-related proteins, such as cytochrome c, PARP, cleaved caspase-3 and caspase-9, Bcl-2 and Bax, using western blotting. Finally, the aforementioned in vitro results were tested in vivo using cell-derived xenograft mouse models, and the anticancer effect was confirmed and attributed to CAP-mediated apoptosis. The immunohistochemical analysis revealed that the expression of cleaved caspase-9, caspase-3, PARP and Bax were upregulated whereas that of Bcl-2 downregulated after CAP treatment. These findings collectively suggest that the activation of the mitochondrial pathway is involved during CAP_PZ2-induced apoptosis of human colon and lung cancer cells in vitro and in vivo.

Keywords: apoptosis; caspase; cold atmospheric plasma; mitochondria; reactive oxygen species.

FIGURE 1

Illustration of the CAP device, Piezobrush^® PZ2, used in this study. (A,B) Content and portability of the CAP_PZ2 kit. (C) Cellular treatment using the handheld plasma device. (D) Close-up of the plasma nozzle in the operating mode.

FIGURE 2

Inhibition of cell proliferation by CAP_PZ2 treatment. (A) Optical images of the HT29 and A549 cellular damage caused by CAP_PZ2 treatment. (B, C) Cell activity decreases while CAP_PZ2 treatment duration increases. The above images and values represent results from three independent experiments and expressed as mean ± SD. *p < 0.05 was considered statistically significant.

FIGURE 3

Effect of CAP_PZ2 treatment on the migratory and invasive ability of the HT29 and A549 cells. The HT29 (A) and A549 (B) cells were treated by CAP_PZ2 for 30 and 60 s, incubated for 24 h, before subjected to Transwell migration and Matrigel invasion assays. Representative images (left) and statistics (right) of migration and invasion assay were shown. The scale bar equals 200 μm. The experiments were performed in triplicates. (C,D) mRNA (MMP9, MMP2, VEGF, MTDH) expression changes during CAP_PZ2 treatment with different time. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control, n = 3.

FIGURE 4

Qualitative analysis of the HT29 and A549 cellular apoptosis upon CAP_PZ2 treatment. (A) Fluorescent microscopic images of nuclear changes (e.g., chromatin condensation) in the HT29 and A549 cells stained with Hoechst33342. (B) Transmission electron microscopic images of ultrathin sections (x10,000) demonstrating subcellular changes in the HT29 and A549 cells treated with CAP_PZ2 for 30 s. All images are at the same magnification.

FIGURE 5

Quantitative analysis of the HT29 and A549 cellular apoptosis upon CAP_PZ2 treatment. (A) Flow cytometric screening of the HT29 and A549 cells treated by CAP_PZ2 for 30 and 60 s. PE Annexin-V/7-ADD staining was used to measure apoptosis rate. The proportion of cells in each quadrant are marked on the figures. (B) Histogram of the corresponding apoptosis rate expressed as mean ± SD of three independent experiments (***p < 0.001 and ****p < 0.0001). (C) Fluorescence microscopic images of the apoptotic HT29 and A549 cells detected using AO/EB staining. Green represents viable cells and orange represents apoptotic cells.

FIGURE 6

Dose-dependent and CAP_PZ2-induced ROS accumulation in the HT29 and A549 cells. (A, B) Flow cytometric analysis of intracellular ROS after DCFH-DA incubation with cells treated using CAP_PZ2 at 30 and 60 s. (C, D) Histogram showing the proportion of ROS negative cells after different plasma treatment duration. Student’s t-test was performed (***p < 0.001 and ****p < 0.0001).

FIGURE 7

Western blot analysis revealing the potential signaling pathway of plasma-induced apoptosis in the HT29 and A549 cells treated using CAP_PZ2. Proteins were extracted from the HT29 (A,B) and A549 (C,D) cells upon treatment by CAP_PZ2 for 30 and 60 s, and subjected to Western blotting using the respective antibodies with β-actin antibody acting as a parallel blotting.*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 versus control, n = 3.

FIGURE 8

In vivo CAP_PZ2 treatment of mouse xenograft tumors. (A) Flowchart of in vivo CAP_PZ2 therapy regimen. (B) Photograph of an operating Piezobrush^® PZ2 CAP device irradiating tumors on a nude mouse bearing the HT29 or A549 cells. (C) Pathological specimens of resected in situ tumors with and without CAP_PZ2 treatment. (D) Tumor growth curves of mice xenografts during the entire course of CAP_PZ2 treatment. (E) Tumor weight after the last dose of plasma administration (*p < 0.05, **p < 0.01, ***p < 0.001, n = 8). (F) Representative histological images characterizing tumours and Immunohistochemical analysis using antibodies against various apoptosis-related proteins. Scale bar = 50 μm.