Canady Helios Cold Plasma Induces Breast Cancer Cell Death by Oxidation of Histone mRNA

Abstract

Breast cancer is the most common cancer among women worldwide. Its molecular receptor marker status and mutational subtypes complicate clinical therapies. Cold atmospheric plasma is a promising adjuvant therapy to selectively combat many cancers, including breast cancer, but not normal tissue; however, the underlying mechanisms remain unexplored. Here, four breast cancer cell lines with different marker status were treated with Canady Helios Cold Plasma™ (CHCP) at various dosages and their differential progress of apoptosis was monitored. Inhibition of cell proliferation, induction of apoptosis, and disruption of the cell cycle were observed. At least 16 histone mRNA types were oxidized and degraded immediately after CHCP treatment by 8-oxoguanine (8-oxoG) modification. The expression of DNA damage response genes was up-regulated 12 h post-treatment, indicating that 8-oxoG modification and degradation of histone mRNA during the early S phase of the cell cycle, rather than DNA damage, is the primary cause of cancer cell death induced by CHCP. Our report demonstrates for the first time that CHCP effectively induces cell death in breast cancer regardless of subtyping, through histone mRNA oxidation and degradation during the early S phase of the cell cycle.

Keywords: breast cancer; cancer treatment; cold atmospheric plasma; histone gene; oxidation of RNA.

Figure 1

Schematic image of Canady Helios Cold Plasma setup for the treatment of breast cancer cells.

Figure 2

Averaged quantification plot of ‘Ki-67⁺’ cell counts of (A) MCF-7, (B) BT-474, (C) MDA-MB-231, and (D) SK-BR-3 cells 6/24/48 h post-CHCP treatment at 120 or 80 P for 3 or 5 min. (* above the bars denotes statistical significance of CAP-treated group compared to NT. * p < 0.05). NT = No Treatment. (E) Representative confocal microscopic images of MCF-7 cells. NT or 6/24/48 h post-CAP treatment at 120 P for 5 min (Alexa Fluor 488 conjugated Ki-67 Rabbit mAb probe was shown in green, DAPI for nucleus staining was shown in blue. Scale bar 20 μm).

Figure 3

Caspase 3/7 activity of breast cancer cell lines at 0–72 h post-CAP treatment (A–D) Quantification of caspase 3/7 activity of MCF-7, BT-474, MDA-MB-231, and SK-BR-3 over 72 h post-CHCP treatment by IncuCyte Live-Cell Imaging. (E–H) Representative images and averaged quantification plots of ‘live’, ‘early apoptosis’, and ‘late apoptosis/dead’ populations of MCF-7, BT-474, MDA-MB-231, and SK-BR-3 at 24 and 48 h post-CHCP treatment by flow cytometry. (* above the bars denotes statistical significance of CAP-treated group compared to corresponding No Treatment group. * p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 4

Quantification of (A) MCF-7, (B) BT-474, and (C) MDA-MB-231 cells in G1, G1-S, S/G2/M, and M-G1 phases over 72 h after CHCP treatment at various dosages.

Figure 5

(A) Heatmap of the top 30 differentially expressed genes between CHCP-treated MDA-MB-231 cells (602) and mocks (605). Gene expression levels are displayed on a log (absolute values) scale. (B) Next-generation sequencing (NGS) whole-transcriptome analysis in fold-changes (log2 scale) of histone and histone-related transcripts after 6 h CHCP treatment in MDA-MB-231 cells. (C) Histone mRNA degradation after CHCP treatment. Fold-change of histone mRNA after 0, 1, 2 and 3 h post-treatment compared to mock control samples of MDA-MB-231 cells.

Figure 6

(A) The fluorescence intensity of RNA oxidation probed in situ with fluorescence-tagged 8-oxoG binding peptide probe on CHCP-treated or untreated live MDA-MB-231 cells. The average SEM of the ratios is plotted. ANOVAs were used followed by post-hoc comparisons using Student’s t-test with Bonferroni corrections as appropriate. *** p < 0.001. (B) Pull-down of 8-oxoG histone RNA; the fold-change of immunoprecipitated 8-oxoG histone RNA after 0 and 1 h post-CAP treatment compared to immunoprecipitated 8-oxoG mock control samples of MDA-MB-231 cells. (C) The percentage change of 8-oxoG modification in histone genes at zero-hour and one-hour incubation after CAP treatment in MDA-MB-231 cells between the CAP-8-oxoG immunoprecipitated group and input group.

Figure 7

Fold-changes in DNA damage response gene transcripts ATF3, CCNE1, EGR1, ID2 and PTGS2 genes compared to mock controls in MDA-MB-231 cells post-CHCP treatment at different incubation time points (3, 6, 12, & 24 h). Statistical analysis was performed using Student’s t-test. * p < 0.005; ** p < 0.001.