Anticancer efficacies of arsenic disulfide through apoptosis induction, cell cycle arrest, and pro-survival signal inhibition in human breast cancer cells

Abstract

Arsenic disulfide, a major effective component of realgar, has been investigated for its anti-cancer potential and shown to have therapeutic efficacies in hematological and some solid tumors. However, its effect against breast cancer is rarely reported. In this study, we investigated the anti-cancer effects of As₂S₂ in human breast cancer cell lines MCF-7 and MDA-MB-231, and further elucidated its underlying mechanisms. As₂S₂ significantly inhibited cell viabilities, induced apoptosis, and led to cell cycle arrest in both cell lines with a dose- and time-dependent manner. As₂S₂ upregulated pro-apoptotic proteins like p53 and PARP in MCF-7 cells. Besides, As₂S₂ downregulated anti-apoptotic proteins like Bcl-2 and Mcl-1, as well as cell cycle-related proteins cyclin A2 and cyclin D1 in both cell lines. Of note, the expression level of cyclin B1 was downregulated in MCF-7 cells, whereas, upregulated in MDA-MB-231 cells. Moreover, As₂S₂ significantly inhibited the pro-survival signals in PI3K/Akt pathway in both cell lines. In conclusion, As₂S₂ inhibited cell viabilities, induced apoptosis and cell cycle arrest in both MCF-7 and MDA-MB-231 cell lines by regulating the expression of key proteins involved in related pathways. These results provide fundamental insights into the clinical application of As₂S₂ for treatment of patients with breast cancer.

Keywords: Arsenic disulfide; MCF-7; MDA-MB-231; apoptosis; cell cycle; cell viability.

Figure 1

Effects of As₂S₂ on the viability of breast cancer cells. (A) MCF-7 and (B) MDA-MB-231 cells were treated with various concentrations of As₂S₂ (0, 4, 8, 12, 16, 20 and 24 µM) for 24 h (●), 48 h (□) and 72 h (▲), respectively, and the cell viability was assessed by CCK-8 assay procedures. All of the data were expressed as the mean ± SEM (n ≥ 3). Asterisks indicate significant differences between the control and the drug treatment groups (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).

Figure 2

Assessment of cell viability by calcein-AM staining. MCF-7 and MDA-MB-231 cells were seeded at the density of 5,000 cells per well. Cells were treated with a serial concentrations of As₂S₂ (0, 4, 8, 12 and 16 µM) for 24, 48 and 72 h, respectively. Viable cells exposed to calcein-AM showed bright green fluorescence. Images were taken and analyzed by a fluorescence Micro-plate reader (Operetta CLS, PerkinElmer, Japan) with 10 × objective (original magnifications 100 ×).

Figure 3

As₂S₂ triggers cell cycle arrest in MCF-7 cells. (A) MCF-7 cells were treated with various concentrations of As₂S₂ (0, 4, 8, 12 and 16 µM) for 24, 48 and 72 h. The peaks in the figure represent the G₀/G₁, S, and G₂/M phases in the cell cycle, respectively. (B) The percentages of cell numbers in the cell cycle of MCF-7 cells after 24 h, (C) 48 h, and (D) 72 h of drug treatment. All data were expressed as the mean ± SEM (n ≥ 3). Asterisks indicate significant differences between the control (0 µM) and the drug treatment groups (*P < 0.05, **P < 0.01).

Figure 4

As₂S₂ triggers cell cycle arrest in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with serial concentrations of As₂S₂ (0, 4, 8, 12 and 16 µM) for 24, 48 and 72 h. The peaks in the figure represent the G₀/G₁, S, and G₂/M phases in the cell cycle, respectively. (B) The percentages of cell numbers in the cell cycle of MDA-MB-231 cells after 24 h, (C) 48 h, and (D) 72 h of drug treatment. All data were expressed as the mean ± SEM (n ≥ 3). Asterisks indicate significant differences between the control (0 µM) and the drug treatment groups (*P < 0.05, **P < 0.01).

Figure 5

Effects of As₂S₂ on cell cycle regulators in MCF-7 cells. MCF-7 cells were cultured with various concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 24, 48 and 72 h, and Western blot assays were carried out to examine the effects of As₂S₂ on the expressions of key proteins cyclin A2, cyclin B1 and cyclin D1 in MCF-7 cells after 24, 48 and 72 h of drug treatment. Protein β-actin was used as internal control. All images are representative of three independent analyses from three independent cellular preparations. Asterisks indicate significant differences between the control (0 µM) and the drug treatment groups (*P < 0.05, **P < 0.01).

Figure 6

Effects of As₂S₂ on cell cycle regulators in MDA-MB-231 cells. MDA-MB-231 cells were cultured with serial concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 24, 48 and 72 h. Western blot assays were carried out to examine the effects of As₂S₂ on the expressions of key proteins cyclin A2, cyclin B1 and cyclin D1 in MDA-MB-231 cells after 24, 48 and 72 h of drug treatment. Protein β-actin was used as internal control. All images are representative of three independent analyses from three independent cellular preparations. Asterisks indicate significant differences between the control (0 µM) and the drug treatment groups (*P < 0.05, **P < 0.01).

Figure 7

As₂S₂ induces apoptosis in breast cancer cells. (A) MCF-7 and (B) MDA-MB-231 cells were treated with different concentrations of As₂S₂ (0, 4, 8, 12 and 16 µM) for 24, 48 and 72 h, followed by staining with Annexin V/PI, and then analyzed by flow cytometry. The cells were assessed for total apoptotic cells composed by early apoptotic (Annexin V⁺/PI^-) and late apoptotic (Annexin V⁺/PI⁺) cells. The apoptotic index was defined as the ratio of total apoptotic cell percentages between As₂S₂ treatment groups (with concentrations of As₂S₂ as 4, 8, 12 and 16 µM) and the control (As₂S₂ as 0 µM). All data were expressed as the mean ± SEM (n ≥ 3). Asterisks indicate significant differences between the control and the drug treatment groups (*P < 0.05, **P < 0.01).

Figure 8

Effects of As₂S₂ on the expression levels of pro-apoptotic proteins in MCF-7 cells. MCF-7 cells were treated with different concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 24, 48 and 72 h. Western blot assays were carried out to examine the effects of As₂S₂ on the expressions of pro-apoptotic proteins p53 and PARP in MCF-7 cells after 24, 48 and 72 h of drug treatment. Protein β-actin was used as internal control. All images are representative of three independent analyses from three independent cellular preparations. Asterisks indicate significant differences between the control (0 µM) and the drug treatment groups (*P < 0.05, **P < 0.01).

Figure 9

Effects of As₂S₂ on the expression levels of pro-apoptotic proteins in MDA-MB-231 cells. MDA-MB-231 cells were treated with different concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 24, 48 and 72 h. Western blot assays were carried out to examine the effects of As₂S₂ on the expressions of pro-apoptotic proteins p53 and PARP in MDA-MB-231 cells after 24, 48 and 72 h of drug treatment. Protein β-actin was used as internal control. All images are representative of three independent analyses from three independent cellular preparations.

Figure 10

Effects of As₂S₂ on the expression levels of anti-apoptotic proteins in MCF-7 cells. MCF-7 cells were treated with different concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 24, 48 and 72 h. Western blot assays were carried out to examine the effects of As₂S₂ on the expressions of anti-apoptotic proteins Bcl-2 and Mcl-1 in MCF-7 cells after 24, 48 and 72 h of drug treatment. Protein β-actin was used as internal control. All images are representative of three independent analyses from three independent cellular preparations. Asterisks indicate significant differences between the control (0 µM) and the drug treatment groups (*P < 0.05, **P < 0.01).

Figure 11

Effects of As₂S₂ on the expression levels of anti-apoptotic proteins in MDA-MB-231 cells. MDA-MB-231 cells were treated with different concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 24, 48 and 72 h. Western blot assays were carried out to examine the effects of As₂S₂ on the expressions of anti-apoptotic proteins Bcl-2 and Mcl-1 in MDA-MB-231 cells after 24, 48 and 72 h of drug treatment. Protein β-actin was used as internal control. All images are representative of three independent analyses from three independent cellular preparations. Asterisks indicate significant differences between the control (0 µM) and the drug treatment groups (*P < 0.05, **P < 0.01).

Figure 12

Effects of As₂S₂ on the expression levels of pro-survival proteins in MCF-7 cells. MCF-7 cells were cultured with various concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 24, 48 and 72 h. Western blot assays were carried out to examine the effects of As₂S₂ on the expressions of key proteins PI3K and Akt in MCF-7 cells after 24, 48 and 72 h of drug treatment. Protein β-actin was used as internal control. All images are representative of three independent analyses from three independent cellular preparations. Asterisks indicate significant differences between the control (0 µM) and the drug treatment groups (*P < 0.05, **P < 0.01).

Figure 13

Effects of As₂S₂ on the expression levels of pro-survival proteins in MDA-MB-231 cells. MDA-MB-231 cells were cultured with serial concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 24, 48 and 72 h. Western blot assays were carried out to examine the effects of As₂S₂ on the expressions of key proteins PI3K and Akt in MDA-MB-231 cells after 24, 48 and 72 h of drug treatment. Protein β-actin was used as internal control. All images are representative of three independent analyses from three independent cellular preparations. Asterisks indicate significant differences between the control (0 µM) and the drug treatment groups (*P < 0.05, **P < 0.01).