BCL2A1 regulates Canady Helios Cold Plasma-induced cell death in triple-negative breast cancer

Abstract

Breast cancer is the leading cause of cancer death among women. Triple-negative breast cancer (TNBC) has a poor prognosis and frequently relapses early compared with other subtypes. The Cold Atmospheric Plasma (CAP) is a promising therapy for prognostically poor breast cancer such as TNBC. The Canady Helios Cold Plasma (CHCP) induces cell death in the TNBC cell line without thermal damage, however, the mechanism of cell death by CAP treatment is ambiguous and the mechanism of resistance to cell death in some subset of cells has not been addressed. We investigate the expression profile of 48 apoptotic and 35 oxidative gene markers after CHCP treatment in six different types of breast cancer cell lines including luminal A (ER⁺ PR^+/-HER2^-), luminal B (ER⁺PR^+/-HER2⁺), (ER^-PR^-HER2⁺), basal-like: ER^-PR^-HER2^- cells were tested with CHCP at different power settings and at 4 different incubation time. The expression levels of the gene markers were determined at 4 different intervals after the treatment. The protein expression of BCL2A1 was only induced after CHCP treatment in TNBC cell lines (p < 0.01), whereas the HER2-positive and ER, PR positive cell lines showed little or no expression of BCL2A1. The BCL2A1 and TNF-alpha expression levels showed a significant correlation within TNBC cell lines (p < 0.01). Silencing BCL2A1 mRNA by siRNA increased the potency of the CHCP treatment. A Combination of CHCP and CPI203, a BET bromodomain inhibitor, and a BCL2A1 antagonist increased the CHCP-induced cell death (p < 0.05). Our results revealed that BCL2A1 is a key gene for resistance during CHCP induced cell death. This resistance in TNBCs could be reversed with a combination of siRNA or BCL2A1 antagonist-CHCP therapy.

Figure 1

CHCP treatment differentially regulates the apoptosis induction genes. Bar graph showing the differential gene expression of genes involved in the induction of apoptosis after CHCP treatment in TNBC cell line MDA-MB-231. The samples were normalized to 18s rRNA and the fold changes were compared to mock controls. Error bars represent means ± SEM. Student t test significant at *p values < 0.05, **p values < 0.01.

Figure 2

CHCP treatment differentially regulates the apoptosis regulation genes. Bar graph showing the differential gene expression of genes involved in the positive (A) and negative (B) regulation of apoptosis after CAP treatment in TNBC cell line MDA-MB-231. The samples were normalized to 18s rRNA and the fold change were compared to mock controls. Error bars represent means ± SEM. Student t test significant at *p values < 0.05, **p values < 0.01.

Figure 3

CHCP treatment differentially regulates Caspase genes. Bar graph showing the differential gene expression of genes involved in the caspase family (A) and caspase activation (B) regulation of apoptosis after CAP treatment in TNBC cell line MDA-MB-231. The samples were normalized to 18s rRNA and the fold changes were compared to mock controls. Error bars represent means ± SEM. Student t test significant at *p values < 0.05, *p values < 0.01.

Figure 4

The regulation of oxidative stress genes APOE was differentially expressed after CHCP treatment. Bar graph showing the differential gene expression of genes involved in the regulation of oxidative stress after CAP treatment in TNBC cell line MDA-MB-231. The samples were normalized to 18s rRNA and the fold changes were compared to mock controls. Error bars represent means ± SEM. Student t test significant at *p values < 0.05, *p values < 0.01.

Figure 5

Differential expression TNF alpha and BCL2A1 are correlated after CHCP treatment. Line graph showing the differential gene expression of TNF (A) and BCL2A1 (B) transcripts after CAP treatment at 120 power for 5 min on TNBC cell line MDA-MB-231. The samples were normalized to 18s rRNA and the fold changes were compared to mock controls. Line graph (C) shows the correlation of BCL2A1 and TNF gene expression after CAP treatment at 120 power for 5 min on two TNBC cell line MDA-MB-231 and Hs578T cells. The samples were normalized to 18s rRNA and the fold changes were compared to mock controls. Error bars represent means ± SEM. Student t test significant at *p values < 0.05, *p values < 0.01.

Figure 6

BCL2A1 protein if only expressed after CHCP treatment in TNBC cells. Expression of BCL2A1 after CAP treatment (CP) and mock controls (M) in TNBC cell line MDA-MB-231. Representative Western blots (A) and bar graph representation (B) of the quantification of BCL2A1 protein in total tissue lysates from MDA-MB-231 cells after CHCP treatment compared to the mock control groups.

Figure 7

Silencing of BCL2A1 mRNA increases CHCP treatment potency. Bar graphs showing the (A) effects of BCL2A1 esiRNA silencing on cell viability and (B) the fold change of BCL2A1 mRNA and (C) protein expression in MDA-MB-231 cell line in combination with CAP treatment. At 24 h. after transfection with BCL2A1 esiRNA (13 pmol), as mentioned in the methods section survival of treatments was determined by MTT assay and BCL2A1 fold change by qRTPCR and western blots. (D) and (E) show a representative blot for BCLA1 and total protein respectively. The error bars represent means ± SEM. (n = 3). (****p < 0.0001, *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test) versus control.

Figure 8

Silencing of BCL2A1 expression increases cell death at G1 phase of the cell cycle after CHCP treatment. (A) Representative pictures of IncuCyte live cell image showing the live state of MDA-MB-231-Cell Cycle Green/Red reagent stable cells treated with/without CHCP treatment or siRNA BCL2A1. The CHCP treated cells recover 50% of the time but when transfected with siRNA-BCL2A1 in combination with CHCP, such recovery is inhibited. (B) Shows the line graph for the quantification of the cells in G1, G1-S, S/G2/M, and M-G1 phases after CHCP treatment corresponding to section A.

Figure 9

Combination of the BET bromodomain inhibitor CPI203 and CHCP treatment increases TNBC cell death. Viability of TNBC cell line MDA-MB-231 with Gambogic acid, an antagonist of antiapoptotic Bcl-2 family (A) or CPI203, a BET bromodomain inhibitor after CAP treatment. Bar graph represents MDA-MB-231 MTT cell viability assay after treatment with Gambogic acid (A) or CPI203 in the combination of with/without CAP treatment at 80p 5 min at 3 L/min He. The samples were compared to the mock control groups. The data is represented by the SEM (n = 3). (*p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test).