Introduction

Oncology Reports

1021-335X 1791-2431

D.A. Spandidos

10.3892/or.2018.6780

or-41-01-0027

Articles

Antitumor effects of arsenic disulfide on the viability, migratory ability, apoptosis and autophagy of breast cancer cells

Zhao

Yuxue

1 2 Onda

Kenji

1 Sugiyama

Kentaro

1 Yuan

3 Tanaka

Sachiko

1 Takagi

Norio

3 Hirano

Toshihiko

1Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan 2Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China 3Department of Applied Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan

Correspondence to: Professor Toshihiko Hirano, Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan, E-mail: hiranot@toyaku.ac.jp

012019

09102018

41 1 27 42 23032018 04092018

2019

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

In the present study, the antitumor effects of arsenic disulfide (As₂S₂) on the proliferative, survival and migratory ability of human breast cancer MCF-7 and MDA-MB-231 cells were investigated, and its potential underlying molecular mechanisms with an emphasis on cell cycle arrest, apoptosis induction, autophagy induction and reactive oxygen species (ROS) generation were determined. The results indicated that As₂S₂ significantly inhibited the viability, survival and migration of breast cancer cells in a dose-dependent manner. In addition, it was identified that As₂S₂ induced cell cycle arrest primarily at G₂/M phase in the two breast cancer cell lines by regulating the expression of associated proteins, including cyclin B1 and cell division cycle protein 2. In addition to cell cycle arrest, As₂S₂ also triggered the induction of apoptosis in cells by activating the expression of pro-apoptotic proteins, including caspase-7 and −8, as well as increasing the B-cell lymphoma 2 (Bcl-2)-associated X protein/Bcl-2 ratio, while decreasing the protein expression of anti-apoptotic B-cell lymphoma extra-large. In addition, As₂S₂ stimulated the accumulation of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II and increased the LC3-II/LC3-I ratio, indicating the occurrence of autophagy. As₂S₂ treatment also inhibited the protein expression of matrix metalloproteinase-9 (MMP-9), but increased the intracellular accumulation of ROS in the two breast cancer cell lines, which may assist in alleviating metastasis and attenuating the progression of breast cancer. Taken together, the results of the present study suggest that As₂S₂ inhibits the progression of human breast cancer cells through the regulation of cell cycle arrest, intrinsic and extrinsic apoptosis, autophagy, MMP-9 signaling and ROS generation.

arsenic disulfide breast cancer cells apoptosis cell cycle autophagy reactive oxygen species

Introduction

Arsenic disulfide (As₂S₂) is an orange-red crystalline mineral and the principal effective component of realgar, and has been used extensively to treat various diseases in ancient China and Europe (1). In recent decades, a series of studies have revealed the marked therapeutic potential of As₂S₂ in hematopoietic tumors, particularly acute promyelocytic leukemia (APL) (2–4). In addition, recent evidence has revealed the potent anticancer effect of As₂S₂ against various human solid cancer cell lines, but with markedly decreased toxicity in normal somatic cells (5–7). Previous studies have demonstrated that As₂S₂ exerts potent anticancer effects in human hepatocellular carcinoma cells, cervical cancer cells, endometrial cancer cells, ovarian cancer cells, malignant melanoma cells, pancreatic carcinoma cells and gastric cancer cells, whereas human normal fibroblast cell lines and other human normal cells, including a lung fibroblast cell line (MRC-5), dermal fibroblast cells (HF and Hs-68), embryonic liver cells (L02) and normal breast epithelial cells (184B5), were much less markedly affected by As₂S₂ treatment (8–12). However, relatively few studies have investigated the potential antitumor activity of As₂S₂ in human breast carcinoma and its underlying molecular mechanisms (12–14).

Breast cancer is one of the most common malignancies among women (15,16). Although conventional therapies, such as chemotherapy and radiotherapy, have improved the outcomes for patients with breast cancer, drug resistance and high rates of recurrence still hamper their efficacies in clinical application (17). Arsenic trioxide (ATO) has been approved by the US Food and Drug Administration in 2000 as an agent for the treatment of APL (18) and reportedly has promising therapeutic potential against breast cancer (19). As₂S₂ has a number of benefits over ATO, including a relatively low toxicity and safety in oral administration, while exerting a similar antitumor effect (20,21). Exploring the antitumor effects of As₂S₂ against breast carcinoma might thus shed new light on the therapeutic potential of this arsenic compound for the treatment of breast cancer.

Programmed cell death (PCD), which refers to any form of cell death mediated by an intracellular death program, serves a fundamental function in biological homeostasis (22,23). Dysregulation of this self-destructive process leads to various human diseases, including breast cancer. Apoptosis (type I cell death) and autophagy (type II cell death) are the two primary forms of PCD defined on the basis of morphological criteria (23,24). Apoptosis, the primary and most well-researched mode of PCD, has been regarded as the principal pathway of PCD (25). Apoptosis induction serves an essential function in anticancer chemotherapies against various types of cancer (26). Autophagy is a highly regulated catabolic process that enables cells to clean up and degrade their own cytoplasmic components (24,27). Autophagy induction is attributed to various stresses that ultimately lead to apoptosis, and organelle dysfunction, metabolic stress, chemotherapies, pathogen infection and starvation are known to induce autophagy (25). There is a complex connection between apoptosis and autophagy; indeed, apoptosis may begin with autophagy, and autophagy may end with apoptosis. It has been suggested that targeting these two self-destructive processes may be a particularly useful chemotherapeutic strategy in the treatment of cancer (28), including breast cancer (29). Accumulating evidence has indicated that apoptosis and autophagy can be induced by As₂S₂ treatment in hematopoietic as well as solid cancer cell lines (8,30,31). Our previous studies revealed the inhibitory effect of As₂S₂ on breast cancer cells, mediated by the induction of apoptosis (12,13). However, the molecular mechanism underlying the involvement of As₂S₂ in apoptosis and autophagy in breast cancer cells remains unclear, warranting further investigation.

Reactive oxygen species (ROS), as a common indicator of oxidative stress, consist of superoxide, hydrogen peroxide and the hydroxyl free radical (32,33). ROS production by xenobiotics selectively kills cancer cells with negligible effects on normal cells (34). Intriguingly, arsenic compounds promote the generation of ROS, and this increased ROS accumulation mediates the genotoxicity of arsenic in cancer cells, thereby facilitating the induction of apoptosis (35–37). ROS therefore serve a pivotal function in cancer cell death caused by arsenic compounds, making them a tempting target for an As₂S₂-based strategy of cytotoxic intervention in breast carcinoma.

The aim of the present study was to investigate the anticancer effects of As₂S₂ in human breast cancer cells in vitro and the potential underlying molecular mechanisms involved, particularly with respect to the induction of PCD and the generation of ROS.

Materials and methods <sec> <title>Reagents

Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Calcein-acetoxymethyl ester (AM) and Hoechst 33342 were purchased from Molecular Probes; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). A Fluorescein Isothiocyanate (FITC)-Phycoerythrin Annexin V Apoptosis Detection kit was obtained from BD Biosciences (San Jose, CA, USA). As₂S₂, propidium iodide (PI), RNase A solution and 2′,7′-dichlorofluorescin diacetate (DCF-DA) were purchased from Sigma; Merck KGaA (Darmstadt, Germany). Chloroquine diphosphate (CQ) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). An Enhanced Chemiluminescence (ECL) Western Blotting Analysis system and ECL Prime Western Blotting Detection reagent were purchased from GE Healthcare Life Sciences (Little Chalfont, UK). Rabbit anti-human matrix metalloproteinase-9 (MMP-9), rabbit anti-human B-cell lymphoma 2 (Bcl-2), rabbit anti-human B-cell lymphoma extra-large (Bcl-xl), rabbit anti-human caspase-7, mouse anti-human caspase-8, rabbit anti-human microtubule-associated protein 1A/1B-light chain 3 (LC3A/B), mouse anti-human cyclin B1 and rabbit anti-human cell division cycle protein 2 (Cdc2) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Mouse anti-human Bcl-2-associated X protein (Bax) was purchased from Sigma; Merck KGaA.

Cell lines and cell culture

The human breast cancer MCF-7 and MDA-MB-231 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in α-minimal essential medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 1% penicillin/streptomycin and fetal bovine serum (10% for MCF-7 and 15% for MDA-MB-231; Sigma; Merck KGaA). The cells were cultured and maintained as attached cells at 37°C in a humidified atmosphere containing 5% CO₂.

Cell culture assays and drug treatment

MCF-7 and MDA-MB-231 cells were seeded at 10,000 and 15,000 cells/well, respectively, in 500 µl cell culture medium on 48-well plates (Iwaki microplates; Iwaki Co., Ltd., Tokyo, Japan), followed by overnight incubation at 37°C. As₂S₂ was subsequently added to the corresponding wells to adjust the final drug concentrations to between 0 and 16 µM. MCF-7 and MDA-MB-231 cells were allowed to grow for 48 h in the presence of different concentrations of As₂S₂, followed by a cytotoxicity assay.

Cytotoxicity assay

Cell cytotoxicity was analyzed using a CCK-8 assay. For each cell line, ~1×10⁴ cells/well were seeded into 48-well plates. As₂S₂ was subsequently added to the corresponding wells to adjust the final drug concentrations to between 0 and 16 µM. The plates were then incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 48 h. Following incubation, 25 µl CCK-8 reagent was added to each well, followed by further incubation at 37°C for 3 h. The optical density (OD) value of each well was determined using a microplate reader (Corona MT P-32; Corona Co., Ibaraki, Japan) at 570 nm. The cell viability rate was calculated according to the following equation: Cell viability rate = (OD sample value - OD blank value)/(OD control value - OD blank value) × 100%.

Morphological analysis and cell viability assay

MCF-7 and MDA-MB-231 cells were seeded onto a 96-well plate at 5×10³ cells/well in 100 µl culture medium, followed by exposure to different concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 48 h. The cells were then stained for 15 min in the dark at 37°C with the specific live probe calcein-AM, prior to capturing images and analysis using an Operetta CLS fluorescence microplate reader (PerkinElmer, Inc., Waltham, MA, USA) and the Harmony software program (version 4.5; PerkinElmer, Inc.).

Wound healing assay

Migration was determined using a wound scratching assay. Cells were seeded at 4×10⁵ cells/well in 6-well plates (Iwaki microplates) and cultured for 24 h to form a confluent cell monolayer. A wound was then scratched onto the cells using a sterile micropipette tip. The cells were washed with PBS and treated with various concentrations of As₂S₂ (0, 8 and 16 µM), followed by further incubation for 48 h. Images of each scratch at the same location were captured at 0 and 48 h using an IX70^® inverted microscope (magnification, ×100) (Olympus Corporation, Tokyo, Japan). Cell migration was quantified by measuring the wound opening area using the ImageJ program (version 1.50i, National Institutes of Health, Bethesda, MD, USA).

Cell cycle analyses

MCF-7 and MDA-MB-231 cells were seeded at 4×10⁵ cells/well in 6-well plates (Iwaki microplates), followed by overnight incubation. Cells were treated with 0, 4, 8 and 16 µM As₂S₂, followed by a further 48 h of incubation at 37°C. Cells were harvested and washed with PBS twice. Cells were fixed in 70% ethanol overnight at −20°C and stained with PI and RNase A solution (5 µg/ml PI and 0.5 µg/µl RNase A). The DNA content was determined by flow cytometry (BD Biosciences), and data were analyzed using the cell cycle analysis software program ModFit LT (version 3.0; Verity Software House, Inc., Topsham, ME, USA).

Morphological characteristics of apoptosis

Hoechst 33342 staining was performed to observe morphological characteristics of apoptotic cells. MCF-7 and MDA-MB-231 cells were seeded onto a 96-well plate at 5×10³ cells/well, followed by exposure to different concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 48 h. The cells were then stained with Hoechst 33342 solution at 37°C for 15 min in the dark. The cells were observed and analyzed for morphological changes of the nucleus using a fluorescence microplate reader and the Harmony software program.

Assessment of apoptosis

MCF-7 and MDA-MB-231 cells were seeded at 2×10⁵ cells/well in 6-well plates (2 ml/well) and treated with serial concentrations of As₂S₂ (0, 4, 8 and 16 µM), followed by additional incubation for 48 h at 37°C. The apoptotic rates for the two cell lines were determined using an FITC-Annexin V Apoptosis Detection kit. The staining procedure was performed according to the manufacturer's protocol. In total, ~1×10⁴ cells were analyzed using a flow cytometer and BD FACSDiva software (version 6.0; BD Biosciences). The cells were subsequently assessed for the total number of apoptotic cells, including early-apoptotic (Annexin V⁺/PI⁻) and late-apoptotic (Annexin V⁺/PI⁺) cells.

Autophagy inhibition in breast cancer cells

To examine whether or not As₂S₂-induced cell death was mediated through autophagy, the autophagy inhibitor CQ (10 µM) was added to MCF-7 and MDA-MB-231 cells 1 h prior to the addition of As₂S₂. Subsequently, As₂S₂ was added at concentrations of 0, 4, 8 and 16 µM. After 48 h of treatment, the CCK-8 assay was performed as aforementioned.

Western blot analyses

The standard Western blot protocol was performed in order to evaluate the protein levels of Bcl-2, Bax, Bcl-xl, caspase-7, caspase-8, cyclin B1, Cdc2 and LC3A/B in MCF-7 and MDA-MB-231 cells. The total protein content was extracted from each cell line treated by As₂S₂ at various final concentrations (0, 4, 8 and 16 µM) for 48 h. In brief, cell lysates were separated by SDS-PAGE (12.5% gel) and transferred onto a polyvinylidene difluoride transfer membrane (Immobilon-P; Merck KGaA). Membranes were blocked with 5% dried skimmed milk powder in Tris-buffered saline containing 0.2% Tween-20 (TBST) for 1 h at room temperature. The membranes were washed with TBST and incubated overnight at 4°C with 1:1,000 anti-rabbit MMP-9 specific antibody (cat. no. 3852; Cell Signaling Technology, Inc.), 1:1,000 anti-rabbit Bcl-2 specific antibody (cat. no. 4223; Cell Signaling Technology, Inc.), 1:1,000 anti-rabbit Bcl-xl specific antibody (cat. no. 2764; Cell Signaling Technology, Inc.), 1:500 anti-mouse Bax specific antibody (cat. no. B8429; Sigma; Merck KGaA), 1:1,000 anti-rabbit caspase-7 specific antibody (cat. no. 12827; Cell Signaling Technology, Inc.), 1:1,000 anti-mouse caspase-8 specific antibody (cat. no. 9746; Cell Signaling Technology, Inc.), 1:1,000 anti-mouse cyclin B1 specific antibody (cat. no. 4135; Cell Signaling Technology, Inc.), 1:1,000 anti-rabbit Cdc2 specific antibody (cat. no. 9112; Cell Signaling Technology, Inc.) and 1:1,000 anti-rabbit LC3A/B specific antibody (cat. no. 12741; Cell Signaling Technology, Inc.). Membranes were also probed with anti-β-actin antibody (cat. no. ab49900; Abcam, Cambridge, UK) at 1:4,000 dilution as the internal control. The membranes were incubated with the aforementioned primary antibodies at 4°C overnight and then incubated with 1:1,000 anti-mouse (cat. no. 7076; Cell Signaling Technology, Inc.) or 1:1,000 anti-rabbit (cat. no. 7074; Cell Signaling Technology, Inc.) specific polyclonal secondary antibodies for 1 h at room temperature, followed by washing three times with TBST. Signals were detected using an ECL Western Blot detection kit in a luminescent image analyzer (LAS-3000; Fujifilm Corporation, Tokyo, Japan).

Determination of ROS

MCF-7 and MDA-MB-231 cells were seeded at 4×10⁵ cells/well in 6-well plates (Iwaki microplates), followed by overnight incubation at 37°C. Cells were treated with different concentrations of As₂S₂ (0, 4, 8 and 16 µM), followed by an additional incubation for 48 h. DCF-DA was then added to the two cell lines to a final concentration of 10 µM and incubated at 37°C for 30 min in the dark. Subsequently, MCF-7 and MDA-MB-231 cells were harvested, washed with PBS and resuspended in 500 µl PBS. The intracellular ROS levels of the two cell lines were detected and analyzed using a flow cytometer and BD FACSDiva software.

Statistical analyses

Statistical analyses were performed using GraphPad Prism software (version 6.0; GraphPad Software, La Jolla, CA, USA). Results are presented as the mean ± standard error of the mean of three or more independent experiments. A one-way analysis of variance followed by Tukey's post hoc test was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Results <sec> <title>As2S2 inhibits the cell viability of breast cancer cells

MCF-7 and MDA-MB-231 cells were cultured in the presence of various concentrations of As₂S₂ ranging between 0 and 16 µM for 48 h, and a CCK-8 assay was performed to determine cell viabilities. As presented in Fig. 1, As₂S₂ inhibited the proliferation of the breast cancer cell lines MCF-7 and MDA-MB-231 in a dose-dependent manner. The half-maximal inhibitory concentrations (IC₅₀ values) of As₂S₂ in MCF-7 and MDA-MB-231 cells were 11.75±1.99 and 8.21±2.07 µM after 48 h of exposure, respectively.

As an additional measurement to monitor cell growth inhibition induced by As₂S₂ in breast cancer cells, the fluorescent dye calcein-AM was used to identify live cells (38). As presented in Figs. 2 and 3, live cell numbers in the two cell lines markedly decreased following treatment with increasing As₂S₂ concentrations in a dose-dependent manner. In MCF-7 cells, compared with the control group (0 µM As₂S₂; 1,232.00±70.74 cells), the live cell number was significantly decreased to 422.00±22.87 (P<0.0001), 291.70±37.17 (P<0.0001) and 85.00±36.76 (P<0.0001) following exposure to 4, 8 and 16 µM As₂S₂ for 48 h, respectively (Fig. 2). In MDA-MB-231 cells, compared with the control group (0 µM As₂S₂; 1,455.00±68.75 cells), the live cell number was significantly decreased to 457.00±84.23 (P<0.0001), 292.80±42.24 (P<0.0001) and 177.00±11.92 (P<0.0001) following exposure to 4, 8 and 16 µM As₂S₂ for 48 h, respectively (Fig. 3).

As2S2 inhibits the motility of breast cancer cells

A scratch assay was performed to assess the effect of As₂S₂ on the motility of breast cancer cells. As presented in Fig. 4, with the dynamic observation at 0 and 48 h after scratching, As₂S₂ treatment significantly inhibited migration of the two cell lines. In MCF-7 cells, compared with the control group (0 µM As₂S₂), the relative migration rates significantly decreased following exposure to 8 µM (P=0.0177) and 16 µM (P=0.0042) As₂S₂. In MDA-MB-231 cells, compared with the control group (0 µM As₂S₂), the relative migration rates significantly decreased following exposure to 8 µM (P<0.0001) and 16 µM (P<0.0001) As₂S₂. These data indicate that As₂S₂ inhibits the motility and invasion of different types of breast cancer cell.

In addition, the expression of the tumor migration- and invasion-associated protein MMP-9 was determined by western blot analysis. As presented in Fig. 5, As₂S₂ treatment significantly decreased MMP-9 expression at concentrations of 8 (P=0.0446) and 16 (P=0.0233) µM in MCF-7 cells in comparison with the control. In contrast, in MDA-MB-231 cells, compared with the control, a statistically significant decrease in the MMP-9 expression occurred at 16 µM As₂S₂ (P=0.0444). These results suggested that As₂S₂ exposure decreased the motility of breast cancer cells due at least in part to its downregulation of MMP-9 signals.

As2S2 triggers cell cycle arrest in breast cancer cells

The effect of As₂S₂ on the cell cycle was assessed by evaluating the proportion of cells in each phase compared with the control in MCF-7 and MDA-MB-231 cells using PI staining and flow cytometry. The results indicated that As₂S₂ mainly induced G₂/M phase arrest in MCF-7 cells and MDA-MB-231 cells (Fig. 6).

In MCF-7 cells, following exposure to As₂S₂ at different concentrations (4, 8 and 16 µM) for 48 h, the proportion of cells in G₂/M phase significantly increased from 4.00±0.75 (0 µM) to 8.81±0.52 (P=0.0003), 9.69±0.06 (P<0.0001) and 12.05±0.31% (P<0.0001), respectively. In MDA-MB-231 cells, As₂S₂ treatment at 4, 8 and 16 µM for 48 h increased the proportion of cells in G₂/M phase from 16.34±0.44 (0 µM) to 22.64±0.33 (P=0.0001), 26.11±0.30 (P<0.0001) and 43.43±1.11% (P<0.0001), respectively, as well as increased the proportion of cells in S phase from 20.40±0.18 (0 µM) to 23.25±0.51 (P=0.0242), 22.94±0.47 (P=0.0451) and 36.81±0.87% (P<0.0001), respectively.

Furthermore, the expression of cell cycle-associated proteins was determined by western blot analysis. As presented in Fig. 7, compared with the control, the expression of cyclin B1 significantly decreased following As₂S₂ treatment at concentrations of 8 (P=0.0014) and 16 (P=0.0002) µM in MCF-7 cells, but increased with As₂S₂ at concentrations of 8 (P=0.0007) and 16 (P=0.0022) µM in MDA-MB-231 cells. Following exposure to As₂S₂ for 48 h, a statistically significant decrease in the expression of Cdc2 occurred in MCF-7 cells treated with 8 µM As₂S₂ (P=0.0348) and in MDA-MB-231 cells treated with 16 µM As₂S₂ (P=0.0028).

These results indicated that As₂S₂ triggers G₂/M phase arrest in MCF-7 and MDA-MB-231 cells by regulating the expression of cell cycle-associated proteins.

As2S2 induces apoptosis in breast cancer cells

Apoptosis induced by As₂S₂ in MCF-7 and MDA-MB-231 was validated using Hoechst 33342 staining and a flow cytometric assay.

As presented in Figs. 2 and 3, the occurrence of typical apoptotic characteristics, such as cell shrinkage, chromatin condensation and nuclei fragmentation (39), was evident in MCF-7 and MDA-MB-231 cells following treatment with As₂S₂ (8 and 16 µM) for 48 h, whereas normal nuclei manifested with a round shape and homogeneous staining.

The induction of apoptosis was investigated further using an Annexin V/PI double-staining assay followed by flow cytometry, which was based on a probe of the total proportion of apoptotic breast cancer cells (Annexin V-positive cells). As presented in Fig. 8, the total proportion of apoptotic MCF-7 cells significantly increased from 1.05±0.48 for the control (0 µM As₂S₂) to 6.35±1.05 (8 µM As₂S₂; P=0.0307) and 19.40±2.03% (16 µM As₂S₂; P<0.0001) following exposure to As₂S₂ for 48 h. In MDA-MB-231 cells (Fig. 7), the proportion of apoptotic cells significantly increased from 3.73±0.32 for the control (0 µM As₂S₂) to 6.57±0.84 (8 µM As₂S₂; P=0.0345) and 15.30±1.40% (16 µM As₂S₂; P<0.0001) following exposure to As₂S₂ for 48 h.

To further confirm the induction of apoptosis by As₂S₂ in breast cancer cells, a western blot analysis was performed to investigate the expression of apoptosis-associated proteins. As presented in Fig. 9, the expression of pro-apoptotic proteins, such as caspase-8 (apoptotic initiator) and −7 (apoptotic executioner), was identified to be increased following treatment with As₂S₂ in a dose-dependent manners in MCF-7 and MDA-MB-231 cells. Compared with the control, the expression of caspase-7 and −8 in MCF-7 cells was significantly increased after 48 h of treatment with As₂S₂ at 8 (P=0.0234 for caspase-7; P=0.0158 for caspase-8) and 16 (P=0.0129 for caspase-7; P=0.0391 for caspase-8) µM. In MDA-MB-231 cells, the expression of caspase-7 and −8 was also significantly increased after 48 h of treatment with As₂S₂ at 16 µM (P=0.0294 for caspase-7; P=0.0018 for caspase-8).

As presented in Fig. 10, the ratio of Bax expression to Bcl-2 expression was increased by As₂S₂ in MCF-7 and MDA-MB-231 cells. Compared with the control, significant increases were observed in MCF-7 cells following treatment with 8 (P=0.0003) and 16 (P<0.0001) µM As₂S₂ for 48 h. In MDA-MB-231 cells, the ratio of Bax to Bcl-2 increased with increasing doses, although the change was not statistically significant. The anti-apoptotic protein Bcl-xl was inhibited by As₂S₂ in MCF-7 and MDA-MB-231 cells. Compared with the control, the expression of Bcl-xl was significantly decreased by As₂S₂ at 16 µM in the two cell lines (P=0.0014 in MCF-7 cells; P=0.0015 in MDA-MB-231 cells).

Taken together, these results indicate that apoptosis was induced by As₂S₂ in MCF-7 and MDA-MB-231 cells in a dose-dependent manner by regulating apoptosis-associated proteins.

As2S2 induces autophagy in breast cancer cells

When autophagy is induced, LC3 is converted from the cytoplasmic form LC3-I into the membrane-associated form LC3-II. The ratio of LC3-II/LC3-I is widely considered as a primary marker of autophagy activation (40,41).

As presented in Fig. 11, As₂S₂ induced an increase in the LC3-II/LC3-I ratio in MCF-7 and MDA-MB-231 cells. Compared with the control, significant increases were observed in MCF-7 cells following treatment with As₂S₂ at 8 (P=0.0007) and 16 (P=0.0187) µM. Similarly, the ratio of LC3-II/LC3-I was increased markedly in MDA-MB-231 cells following treatment with As₂S₂ at 8 (P=0.0048) and 16 (P=0.0358) µM.

Autophagy has been identified to serve functions in cytoprotective and cytotoxic processes. To examine the effect of As₂S₂-induced autophagy on breast cancer cell viability, MCF-7 and MDA-MB-231 cells were exposed to the autophagy inhibitor CQ in the presence of different concentrations of As₂S₂ (0, 4, 8 and 16 µM). As presented in Fig. 12A, inhibition of autophagy did not alter the inhibitory effect of As₂S₂ on MCF-7 cells. Furthermore, although pretreatment with CQ significantly reversed the cell death induced by As₂S₂ at 4 µM (P=0.0021) in MDA-MB-231 cells, no additional effect was observed in the presence of As₂S₂ treatment at 8 or 16 µM (Fig. 12B).

Effects of As2S2 on ROS production in breast cancer cells

The effects of As₂S₂ on ROS production in breast cancer cells were assessed using an ROS-sensitive probe, DCF-DA, and a flow cytometric assay.

As presented in Fig. 13, As₂S₂ induced the accumulation of ROS in a dose-dependent manner in the two breast cancer cell lines. The proportions of ROS production significantly increased from 23.73±1.78 in the control to 29.60±1.20 (P=0.0353) and 33.33±0.32% (P=0.0022) by As₂S₂ at 8 and 16 µM in MCF-7 cells, respectively. In MDA-MB-231 cells, As₂S₂ significantly increased the proportions of ROS production from 6.80±0.06% in the control to 22.30±1.72 (P=0.0001), 27.10±1.20 (P<0.0001) and 28.30±1.43% (P<0.0001) at As₂S₂ concentrations of 4, 8 and 16 µM, respectively.

Discussion

Previous studies have identified the antitumor effect of As₂S₂ against hematopoietic and solid cancer cell lines (5–7,20,21); however, few have investigated the effects of As₂S₂ against breast cancer cell lines. In the present study, the anticancer effect of As₂S₂ on the proliferation and migration of two distinctive subtypes of breast carcinoma cells (MCF-7 and MDA-MB-231) and the molecular mechanisms underlying these effects were investigated, particularly with regard to the activation of PCD. To the best of our knowledge, the present study is the first focusing on the two classical PCD pathways, i.e. apoptosis and autophagy, induced by As₂S₂ in human breast cancer cell lines.

Tumor metastasis, the most deadly aspect of cancer, is a multistep aggressive process involving cell proliferation and cell migration (42,43). In the present study, the antitumor effects of As₂S₂ against the development of breast cancer cells, including cell proliferation, survival and migration, were investigated. Results obtained from a CCK-8 assay indicated that As₂S₂ significantly inhibited the cell viabilities in the two cell lines in a dose-dependent manner. Consistent with these results, the data visualized and obtained using the calcein-AM staining test showed the potent induction of cell death by As₂S₂ in the two cell lines. In addition, the results from wound healing assays indicated a significant decrease in cell invasion by treatment with As₂S₂ in a dose-dependent manner in the two cell lines, which further suggested the tumor suppressive effects of As₂S₂ by repressing the proliferative and migratory abilities of breast carcinoma cells. MMPs are associated with extracellular matrix degradation, which leads to cancer cell invasion and metastasis. MMP-9 is a key factor that contributes to the metastatic potential and cancer progression (44). The migration and invasion of cancer cells are facilitated by MMP-9. Thus, downregulating MMP-9 may be a possible strategy for attenuating the progression of cancer cells (45). The results of the present study indicated that As₂S₂ decreased the protein expression of MMP-9 in the two breast cancer cell lines in a dose-dependent manner, which may account for the decrease in migration in the two cell lines following As₂S₂ treatment.

Cell cycle dysfunction is a common feature of proliferating and metastatic breast tumor cells (46); targeting cell cycle regulation is therefore an important therapeutic strategy. In the present study, it was demonstrated that As₂S₂ treatment exerted pronounced cell cycle arrest at G₂/M phase in the two breast cancer cell lines in a dose-dependent manner. In addition, As₂S₂ triggered G₀/G₁-phase arrest in MCF-7 cells, but S-phase arrest in MDA-MB-231 cells. The exertion of arrest at different phases between the two cell lines is likely to be due to characteristics specific to these cells as distinct subtypes of breast carcinoma. Considering the observed cell cycle arrest, the results of the present study suggest that As₂S₂ treatment regulates the expression of cell cycle-associated proteins involved in the corresponding phases of the cell cycle in each of the breast cancer cell lines. For example, as the main regulatory proteins in G₂/M phase (47,48), the expression of cyclin B1 and Cdc2 was regulated by As₂S₂ treatment in the two cell lines, which is consistent with the blockage of the cell cycle at G₂/M by As₂S₂ treatment. Intriguingly, the protein expression of cyclin B1 was regulated by As₂S₂ in an opposite manner between MCF-7 and MDA-MB-231 cells, possibly due to biological variations and distinctions between these two cell lines. In addition to regulating G₂/M phase, Cdc2 has also been described as a key regulator associated with G₀/G₁ and S phases (38,39), which accounts for the cell cycle arrest observed at G₀/G₁ phase in MCF-7 cells and at the S phase in MDA-MB-231 cells.

Apoptosis and autophagy are two well-known PCD mechanisms that serve essential functions in maintaining organismal and cellular homeostasis (49). Apoptosis, regarded as a major mechanism of chemotherapy-induced cell death (50), is characterized by typical morphological changes, such as nuclear condensation and fragmentation. In the present study, apoptosis was significantly induced by As₂S₂ treatment in the two breast cancer cell lines, as demonstrated using the Annexin V staining assay and visualized using the Hoechst 33342 staining assay. In addition, the induction of apoptosis in the two breast cancer cell lines was further confirmed by the activation of caspase-8 and −7, as well as the regulation of proteins in the Bcl-2 family, which resulted in an increase in the Bax/Bcl-2 ratio along with the decreased expression of Bcl-xl. An essential step in triggering apoptosis is the activation of caspases, a family of cysteine proteases that are ubiquitously expressed as death proteases (23,51). The caspase family has traditionally been divided into initiator and effector caspases. Activated initiator caspases, such as caspase-8, −9 and −10, subsequently initiate a caspase cascade of downstream effector caspases (52). Effector caspases, such as caspase-3, −6 and −7, are understood to execute apoptosis following being triggered by initiator caspases (52). Two principal signaling pathways exist to induce cell apoptosis: The extrinsic (cell death receptor) pathway and the intrinsic (mitochondrial) pathway (53). Of note, caspase-8 has been regarded as a core initiating component of the extrinsic pathway, subsequently activating the execution phase of apoptosis (e.g. activating effector caspase-7) (54). Bcl-2 family proteins are categorized into subgroups on the basis of their pro- or anti-apoptotic actions: Pro-apoptotic proteins, such as Bax, and anti-apoptotic proteins, such as Bcl-2 and Bcl-xl. These Bcl-2 family proteins serve an important function in initiating the intrinsic apoptotic pathway (55,56). In the present study, the expression of caspase-8 and the Bax/Bcl-2 ratio were significantly increased, whereas the expression of Bcl-xl was markedly decreased following As₂S₂ treatment, suggesting that the extrinsic and intrinsic pathways were involved in As₂S₂-induced apoptosis in the two breast cancer cell lines.

Apoptosis and autophagy normally occur in the same cell, primarily in a mutually interactive manner under the same cellular conditions (24,49). Autophagy, a catabolic process for the degradation of unnecessary and dysfunctional cytosolic components and organelles, has generally been regarded as the type II (non-apoptotic) PCD and is deemed an important mechanism involved in the tumor control process (57,58). LC3 is a key protein involved in initiating autophagy, wherein LC3-I is lipidated and converted into LC3-II. The ratio of LC3-II/LC3-I is widely used as a primary marker of autophagy activation (41). The results of the present study indicated that As₂S₂ treatment induced an increase in the ratio of LC3-II/LC3-I in the two breast cancer cell lines, suggesting that autophagy occurs in breast cancer cells following exposure to As₂S₂. Bcl-2 is an intermediary protein shared by apoptosis and autophagy, serving an anti-apoptotic and anti-autophagy function in the two processes (29,59). The results of the present study indicated that As₂S₂ treatment significantly decreased the protein expression of the anti-apoptotic Bcl-2, which may in turn potentiate the induction of autophagy. Intriguingly, autophagy is commonly regarded as a double-edged sword (60,61) and may positively or negatively influence cancer cell growth (62). To improve understanding of the function of autophagy in the present study, CQ, a pharmacological inhibitor of autophagy, was used to clarify whether or not As₂S₂-induced cell death was associated with the induction of autophagy. The results indicated that CQ significantly reversed the inhibitory effect of As₂S₂ (4 µM) on the cell viability in MDA-MB-231 cells, indicating that autophagy induced by As₂S₂ at a relatively low concentration was indeed involved in the death of MDA-MB-231 cells. In contrast, CQ had little effect on the death of MCF-7 and MDA-MB-231 cells in the presence of relatively high concentrations (8 and 16 µM) of As₂S₂, suggesting that the induction of cell death is primarily through apoptosis and cell cycle arrest, as indicated by the results of the present study, rather than autophagy.

ROS help to regulate a series of biological processes, including PCD (63). It has been identified that the accumulation of ROS is associated with cell apoptosis, cell cycle arrest and autophagy induced by anticancer agents, which consequently leads to negative effects on the cell survival, proliferation and metastasis (44,64–67). In the present study, results from flow cytometric analyses indicated that the ROS level in MCF-7 and MDA-MB-231 cells significantly increased following As₂S₂ treatment in a dose-dependent manner, which may potentiate the induction of apoptosis, cell cycle arrest and autophagy in the two cell lines, triggered by As₂S₂ treatment. Accordingly, the oxidative stress generated by ROS production and the activated PCD pathway markedly inhibited cell viability, decreased the live cell number, suppressed cell viability, attenuated cell migration and consequently decreased cell progression in MCF-7 and MDA-MB-231 cells. The potential molecular mechanisms underlying the inhibitory effect of As₂S₂ on breast carcinoma progression are presented in Fig. 14.

In conclusion, the results of the present study identified the antitumor effects of As₂S₂ against breast carcinoma in vitro as inhibition of cell viability, decreased cell survival and attenuated invasion of MCF-7 and MDA-MB-231 cells. These effects were associated with inhibition of cell cycle progression, the induction of apoptosis and autophagy, a decrease in MMP-9 expression and an increase in ROS accumulation. In future studies, we intend to investigate the therapeutic potential of As₂S₂ in vivo in the treatment of breast cancer in animal models.

Acknowledgements

Not applicable.

Funding

The present study was supported in part by the China Scholarship Council (grant no. 201709110064).

Availability of data and materials

The analyzed datasets generated during the study are available from the corresponding author on reasonable request, while preserving the necessary confidentiality and anonymity.

Authors' contributions

TH conceived and designed the study and critically revised the manuscript. YZ designed and performed the experiments, analyzed the data and was a major contributor in writing the manuscript. KO, KS, BY, ST and NT gave advice on the experiments and contributed with reagents and technical assistance. TH and NT supervised the study. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Abbreviations

ATO

arsenic trioxide

APL

acute promyelocytic leukemia

PCD

programmed cell death

CCK-8

Cell Counting Kit-8

propidium iodide

chloroquine diphosphate

MMP-9

matrix metalloproteinase-9

Bcl-2

B-cell lymphoma 2

Bcl-xl

B-cell lymphoma extra-large

Bax

Bcl-2-associated X protein

Cdc2

cell division cycle protein 2

LC3

microtubule-associated protein 1A/1B-light chain 3

DCF-DA

2′,7′-dichlorofluorescin diacetate

ROS

reactive oxygen species

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Figure 1.

As₂S₂ inhibits the viability of breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with various concentrations (0, 4, 8, 12 and 16 µM) of As₂S₂ for 48 h, and the cell viability was determined using Cell Counting Kit-8 assays. Results are presented as the mean ± standard error of the mean (n≥3). *P<0.05, **P<0.01 vs. control group (0 µM As₂S₂).

Figure 2.

As₂S₂ induces changes in calcein-AM and Hoechst 33342 staining in MCF-7 cells. MCF-7 cells were seeded at 5,000 cells/well. The cells were treated with serial concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 48 h. (A) Viable cells exposed to calcein-AM exhibited bright green fluorescence. Hoechst 33342 staining, as a nuclear counterstain, exhibited bright blue fluorescence. Cells with bright fragmented or condensed nuclei (arrows) were identified as those undergoing apoptosis. Merging of calcein-AM- and Hoechst 33342-stained cells exhibited cyan fluorescence. Images were captured and analyzed using a fluorescence microplate reader with a ×20 objective (original magnification, ×200). (B) Quantitative analysis of live MCF-7 cells. Results are presented as the mean ± standard error of the mean (n≥3). **P<0.01 vs. control group (0 µM As₂S₂). AM, acetoxymethyl ester.

Figure 3.

As₂S₂ induces changes in calcein-AM and Hoechst 33342 staining in MDA-MB-231 cells. MDA-MB-231 cells were seeded at 5,000 cells/well. The cells were treated with serial concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 48 h. (A) Viable cells exposed to calcein-AM exhibited bright green fluorescence. Hoechst 33342 staining as a nuclear counterstain exhibited bright blue fluorescence. Cells with bright fragmented or condensed nuclei (arrows) were identified as those undergoing apoptosis. Merging of calcein-AM- and Hoechst 33332-stained cells exhibited cyan fluorescence. Images were captured and analyzed using a fluorescence microplate reader with a ×20 objective (original magnification, ×200). (B) Quantitative analysis of live MDA-MB-231 cells. Results are presented as the mean ± standard error of the mean (n≥3). **P<0.01 vs. control group (0 µM As₂S₂). AM, acetoxymethyl ester.

Figure 4.

Inhibitory effects of As₂S₂ on the migration of MCF-7 and MDA-MB-231 cells determined using a wound healing assay. MCF-7 and MDA-MB-231 cells were treated with various concentrations (0, 8 and 16 µM) of As₂S₂ for 48 h, and then the wound areas were observed. (A) Representative images of wounded MCF-7 cells (magnification, ×100). (B) Quantification of relative migration of MCF-7 cells. (C) Representative images of wounded MDA-MB-341 cells (magnification, ×100). (D) Quantification of relative migration of MDA-MB-341 cells. Results are presented as the mean ± standard error of the mean (n≥3). *P<0.05, **P<0.01 vs. control (0 µM As₂S₂).

Figure 5.

Effects of As₂S₂ on the protein expression of MMP-9 in breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with different concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 48 h. Western blot assays were performed to examine the effects of As₂S₂ on the expression of MMP-9 in the two cell lines after 48 h of treatment. β-actin was used as an internal control. All images are representative of three independent analyses from three independent cellular preparations. *P<0.05 vs. control (0 µM As₂S₂).

Figure 6.

As₂S₂ triggers cell cycle arrest in breast cancer cells. (A) MCF-7 and (B) MDA-MB-231 cells were treated with various concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 48 h. The peaks represent G₀/G₁, S and G₂/M phases in the cell cycle. Quantification of the proportions of cells in a given phase of the cell cycle in (C) MCF-7 and (D) MDA-MB-231 cells after 48 h of treatment. Results are expressed as the mean ± standard error of the mean (n≥3). *P<0.05, **P<0.01 vs. control (0 µM As₂S₂).

Figure 7.

Effects of As₂S₂ on cell cycle regulators in breast cancer cells. MCF-7 and MDA-MB-231 cells were cultured with various concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 48 h, and western blot assays were performed to examine the effects of As₂S₂ on the expression of the key proteins cyclin B1 and Cdc2 in the two cell lines after 48 h of treatment. β-actin was used as an internal control. All images are representative of three independent analyses from three independent cellular preparations. *P<0.05, **P<0.01 vs. control (0 µM As₂S₂).

Figure 8.

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 and 16 µM) for 48 h, followed by staining with Annexin V/PI, and then analyzed by flow cytometry. The cells were assessed for the total number of apoptotic cells, including early-apoptotic (Annexin V⁺/PI⁻) and late-apoptotic (Annexin V⁺/PI⁺) cells. Results are expressed as the mean ± standard error of the mean (n≥3). *P<0.05, **P<0.01 vs. control (0 µM As₂S₂). PI, propidium iodide; PE, phycoerythrin.

Figure 9.

Effects of As₂S₂ on the protein expression of caspase in breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with different concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 48 h. Western blot assays were performed to examine the effects of As₂S₂ on the expression of caspase-7 and −8 in the two cell lines after 48 h of treatment. β-actin was used as an internal control. All images are representative of three independent analyses from three independent cellular preparations. *P<0.05, **P<0.01 vs. control (0 µM As₂S₂).

Figure 10.

Effects of As₂S₂ on the expression of Bcl-2 family proteins in breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with different concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 48 h. Western blot assays were performed to determine the effects of As₂S₂ on the expression of Bax, Bcl-2 and Bcl-xl in the two cell lines after 48 h of treatment. β-actin was used as an internal control. All images are representative of three independent analyses from three independent cellular preparations. **P<0.01 vs. control (0 µM As₂S₂). Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Bcl-xl, B-cell lymphoma extra-large.

Figure 11.

Effects of As₂S₂ on the expression of autophagy hallmarks in breast cancer cells. MCF-7 cells were treated with different concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 48 h. Western blot assays were performed to examine the effects of As₂S₂ on the expression of the autophagy markers LC3-I and LC3-II in the two cell lines after 48 h of treatment. The ratio of LC3-II to LC3-I was calculated to determine the autophagic level. β-actin was used as an internal control. All images are representative of three independent analyses from three independent cellular preparations. *P<0.05, **P<0.01 vs. control (0 µM As₂S₂). LC3, microtubule-associated protein 1A/1B-light chain 3.

Figure 12.

Cell viability in breast cancer cells following autophagy inhibition and As₂S₂ treatment. (A) MCF-7 and (B) MDA-MB-231 cells were pretreated with 10 µM CQ for 1 h before treatment with various concentrations (0, 4, 8 and 16 µM) of As₂S₂ for 48 h. Cell viability was determined using Cell Counting Kit-8 assays. Results are presented as the mean ± standard error of the mean (n≥3). *P<0.05, **P<0.01 vs. control (0 µM As₂S₂ and 0 µM CQ); ^††P<0.01 vs. control (0 µM As₂S₂ and 10 µM CQ); ^##P<0.01 vs. respective As₂S₂ treatment groups in the absence of CQ. CQ, chloroquine diphosphate.

Figure 13.

Effects of As₂S₂ on ROS production in breast cancer cells. (A) MCF-7 and (B) MDA-MB-231 cells were treated with different concentrations of As₂S₂ (0, 4, 8 and 16 µM) for 48 h. Intracellular ROS levels were analyzed using the ROS-responsive dye 2′,7′-dichlorofluorescin diacetate followed by a flow cytometric assay. Results are presented as the mean ± standard error of the mean (n≥3). *P<0.05, **P<0.01 vs. control (0 µM As₂S₂).

Figure 14.

Schematic diagram of the potential molecular mechanisms underlying As₂S₂-hampered carcinoma progression in MCF-7 and MDA-MB-231 cells. ROS, reactive oxygen species; MMP-9, matrix metalloproteinase 9; cdc2, cell division cycle 2.