Tetraarsenictetrasulfide and Arsenic Trioxide Exert Synergistic Effects on Induction of Apoptosis and Differentiation in Acute Promyelocytic Leukemia Cells

Abstract

Since arsenic trioxide (As³⁺) has been successfully used in the treatment of acute promyelocytic leukemia (APL), its adverse effects on patients have been problematic and required a solution. Considering the good therapeutic potency and low toxicity of tetraarsenictetrasulfide (As₄S₄) in the treatment of APL, we investigated the effects of combining As₄S₄ and As³⁺ on the apoptosis and differentiation of NB4 and primary APL cells. As₄S₄, acting similarly to As³⁺, arrested the G₁/S transition, induced the accumulation of cellular reactive oxygen species, and promoted apoptosis. Additionally, low concentrations of As₄S₄ (0.1–0.4 μM) induced differentiation of NB4 and primary APL cells. Compared with the As₄S₄- or As³⁺-treated groups, the combination of As₄S₄ and As³⁺ obviously promoted apoptosis and differentiation of NB4 and primary APL cells. Mechanistic studies suggested that As₄S₄ acted synergistically with As³⁺ to down-regulate Bcl-2 and nuclear factor-κB expression, up-regulate Bax and p53 expression, and induce activation of caspase-12 and caspase-3. Moreover, the combination of low concentrations of As₄S₄ and As³⁺ enhanced degradation of the promyelocytic leukemia-retinoic acid receptor α oncoprotein. In summary, As₄S₄ and As³⁺ synergistically induce the apoptosis and differentiation of NB4 and primary APL cells.

Introduction

Acute promyelocytic leukemia (APL) is an M3 subtype of acute myeloid leukemia [1]. The typical characteristic of APL is the specific chromosomal translocation t(15;17) (q22;q21), which induces the expression of the promyelocytic leukemia-retinoic acid receptor α (PML-RARα) oncoprotein [1–3]. Two drugs, all-trans retinoic acid and arsenic trioxide (As³⁺), have hitherto been successfully used in the treatment of APL [4–6]. At high concentrations (0.5–2.0 μM), As³⁺ triggers apoptosis, and at low concentrations (0.1–0.5μM) it induces partial differentiation of APL cells [7]. Mechanistic studies have suggested that As³⁺ promotes apoptosis and differentiation of APL cells by inducing degradation of the PML-RARα oncoprotein [8]. However, As³⁺ organ injury, especially to the liver and kidneys, causes significant pain to patients [9,10]. Methylation of As³⁺ can induce the accumulation of reactive oxygen species (ROS) and generate more toxic monomethylarsonous and dimethylarsinous acids [11–14]. Currently, combination therapy is widely used in cancer treatment. Therefore, combination therapy for APL treatment can enhance As³⁺ therapeutic potency and reduce its adverse effects.

In addition to As³⁺, realgar is another inorganic form of arsenic that has been used in traditional Chinese medicine for many years [15,16]. Compared with As³⁺, realgar has a positive therapeutic reputation and reduced toxicity [17]. Lu et al. have reported that when used alone, the major constituent of realgar, tetraarsenic tetrasulfide (As₄S₄), showed high efficiency and safety in all stages of APL [18]. Wang et al. showed that the combination of As₄S₄, indigo and naturalis can promote the differentiation of APL cells, induce degradation of the PML-RARα fusion protein, and arrest the cell cycle at G₁/G₀ [19]. However, the molecular mechanism of As₄S₄ potency in APL treatment is unclear. Moreover, realgar is a mixture that contains up to approximately 10% trivalent arsenicals [18], and both bivalent and trivalent arsenicals may contribute to the therapeutic potency of realgar. Clarifying the mechanism of action of As₄S₄ and As³⁺ combination on the apoptosis and differentiation of APL cells is necessary.

Apoptosis is the major pathway for drug-induced cancer cell death [20]. Mitochondria-mediated intrinsic apoptosis, death receptor-mediated extrinsic apoptosis and endoplasmic reticulum stress-mediated apoptosis are the three predominant apoptosis pathways and are regulated by a series of apoptotic factors [21]. Of these factors, Bcl-2 family members [22], nuclear factor-κB (NFκB) [23], p53 tumor suppressor [24], caspase-12 and caspase-3 play key roles in As³⁺-induced apoptosis [25]. Although the function of PML is unclear, degradation of the PML-RARα oncoprotein contributes to apoptosis and differentiation of APL cells [8,26]. These apoptotic factors, as well as the PML-RARα fusion protein, may be important for clarifying the mechanism of As₄S₄-induced apoptosis and differentiation in APL cells.

The NB4 cell line is a unique APL-derived cell line that expresses the PML-RARα oncoprotein [27]. In this work, we found that As₄S₄ and As³⁺ exerted synergistic effects on the apoptosis and differentiation of NB4 and primary APL cells. Multiple pathways were involved in As₄S₄ and As³⁺-induced apoptosis. As₄S₄ and As³⁺ acted synergistically to promote apoptosis of NB4 cells by up-regulating p53 expression, enhancing the mitochondria-mediated intrinsic pathway, enhancing the endoplasmic reticulum stress-mediated pathway, and inhibiting the NFκB signaling pathway. Moreover, low doses of As₄S₄ could be combined with As³⁺ to enhance degradation of the PML-RARα oncoprotein and promote NB4 and primary APL cell differentiation through the retinoic acid-signaling pathway.

Materials and Methods

Caution: Due to the potential risk of arsenic compounds, safeguards should be implemented [11–14].

Reagents

High purity As₄S₄ was obtained from Yiji industry (Shanghai, China). NaAsO₂, bovine serum albumin (BSA), anti-PML rabbit mAb and anti-caspase-12 rabbit mAb were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Bcl-2 (50E3) rabbit mAb, anti-Bax (D2E11) rabbit mAb, anti-NFκB p65 (D14E12) XP rabbit mAb, anti-caspase-3 (8G10) rabbit mAb and anti-p53 (7F5) rabbit mAb were obtained from Cell Signaling Technology (Boston, MA, USA). Anti-β-actin mouse mAb was purchased from Beyotime (Nantong, China). FITC anti-human CD11b antibody was obtained from BioLegend (San Diego, CA. USA).

Cell culture and growth assay

Human NB4 leukemia cells were purchased from SXBIO Biotech (Shanghai, China). Human primary APL cells were separated from the bone marrow of four primary APL patients acquired from DrumTower Hospital (Nanjing, China) by Ficoll-Hypaque density centrifugation as reported in the previously published article [28]. Written informed consent was obtained from individual subjects. For this case we did not seek approval of the Ethics Committee of Drum Tower Hospital and did not obtain a waiver from the Ethics Committee because the bone marrow was part of that acquired for clinical diagnosis and destroyed after this experiment. NB4 cells were cultured in RPMI-1640 (KeyGEN Biotech, China) with 10% fetal bovine serum (FBS) at 37°C under a 5% CO₂ atmosphere. The fresh primary APL cells were cultured in RPMI-1640 with 15% FBS. Trypan blue exclusion was used to determine viability of NB4 and primary APL cells after 48 h and 96 h of culture [28]. The effects of As₄S₄ and As³⁺ on cell growth were determined using the WST-1 cell proliferation assay kit (KeyGEN Biotech, China). In brief, 4×10⁴ cells/ml were seeded into a 96-well culture plate and treated with various concentrations of As₄S₄, As³⁺, or their combination for 48 h. Untreated cells served as controls [28,29].

Calculation of combination index (CI)

The CI values were calculated by equation (1): CI = [D]₁/[D_x]₁ + [D]₂/[D_x]₂ + α[D]₁[D]₂/[D_x]₂[D_x]₂ [30]. Here, [D_x]₁ and [D_x]₂ respectively represent the concentrations of As³⁺ and As₄S₄ alone, resulting in growth inhibition of NB4 and primary APL cells (in x%). [D]₁ and [D]₂ are the concentrations of As³⁺ and As₄S₄ when they are used in combination to inhibit the cell growth at same percentage (x%). When the two drugs are assumed to be non-exclusive, the value of α is 1; when the two drugs are assumed to be exclusive, the value of α is 0. CI = 1 indicate an additive effect; CI < 1indicate a synergistic effect; CI > 1 indicate an antagonism effect [30].

Analysis of apoptosis

The proportions of apoptotic cells were measured with the Annexin V-FITC and propidium iodide (PI) apoptosis detection kit (KeyGEN Biotech, China) using flow cytometry [28,29,31]. NB4 and primary APL cells were treated with 2.0 μM As₄S₄, 2.0 μM As³⁺, or 1.0 μM As₄S₄ and 1.0 μM As³⁺ for 48 h. After treatment, the cells were washed twice with Ca²⁺-free phosphate buffer (PBS), stained with Annexin V-FITC and PI in the dark at room temperature for 15 min, and detected using a BD LSRL Fortessa flow cytometer. The percentages of apoptotic cells were analyzed using the BD FACSDiva software.

Analysis of cell cycle distribution

The effects of As₄S₄ and As³⁺ on cell cycle distribution were determined using a PI cell cycle detection kit (KeyGEN Biotech, China) [28]. After treatment with As₄S₄ (2.0 μM), As³⁺ (2.0 μM), or a combination of 1.0 μM As₄S₄ and 1.0 μM As³⁺ for 48 h, NB4 and primary APL cells were collected, washed with Ca²⁺-free PBS and fixed with 70% ethanol at 4°C for 16 h. The fixed cells were then digested in PBS containing 0.5 mg/ml RNase (Sigma-Aldrich, USA) at 37°C for 30 min and stained with 0.05 mg/ml PI in the dark at room temperature for 30 min. Data on the cell cycle distribution were determined using the ModFit LT 3.3 software.

Analysis of cellular ROS levels using 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA)

Cellular ROS levels were assayed using DCFH-DA. DCFH-DA can be hydrolyzed to DCFH by esterases and oxidized to 2’,7’-dichlorofluorescein (DCF) by cellular ROS [32]. Thus, the DCF fluorescence intensity is positively correlated with cellular ROS levels. After treatment with As₄S₄ (0.5, 1.0 and 2.0 μM), As³⁺ (2.0 μM), or a combination of 1.0 μM As₄S₄ and 1.0 μM As³⁺ for 24 h and 36 h, NB4 and primary APL cells were washed twice with PBS and then incubated in RPMI-1640 medium containing 10.0 μM DCFH-DA at 37°C for 30 min. Excess probe was washed out with PBS, and the percentages of DCF-positive cells were detected by flow cytometry and analyzed using FlowJo.7.6.

Analysis of mRNA by RT-PCR

RNAiso Plus (Takara-Bio, Japan) was used to extract total RNA from NB4 and primary APL cells. The concentration and purity of the isolated total RNA were determined by trace nucleic acid protein measurement instrument (NanoDrop ND-1000) [28,29]. 2.0 μg of total RNA was reverse-transcribed to cDNA, and 2.0 μL of transcribed cDNA was used for PCR amplification with specific primers. After initial denaturing at 94°C for 5 min, thirty cycles of 30 s denaturation at 94°C; 30 s annealing at 52°C (β-actin), 57°C (heme oxygenase-1 (HMOX1), Bax, Bcl-2, and NFκB-3), or 51°C (caspase-3); and 30 s extension at 72°C were performed. The PCR products were separated on 1% agarose gels containing ethidium bromide. The separated bands were imaged on a Gel Doc XR System (Bio-Rad). The primer sequences are shown in Table 1.

Table 1. Primer sequences for the apoptosis factors in RT-PCR.

Name	Primer sequence
HMOX1	sense	5′-CTTTGAGGAGTTGCAGGAGC-3′
	antisense	5′-TGTAAGGACCCATCGGAGAA-3′
Bax	sense	5′-TGACGGCAACTTCAACTGGG-3′
	antisense	5′-AGCACTCCCGCCACAAAGA-3′
Bcl-2	sense	5′-GGGAGGATTGTGGCCTTCTT-3′
	antisense	5′-GGCCAAACTGAGCAGAGTCTTC-3′
NFκB-3	sense	5′-ACTACGAGGGACCAGCCAAGA-3′
	antisense	5′-CGCAGCCGCACTATACTCA-3′
Caspase-3	sense	5′-GTGGAATTGATGCGTGATG-3′
	antisense	5′-AACCAGGTGCTGTGGAGTA-3′
β-Actin	sense	5′-GACCTGACTGACTACCTC-3′
	antisense	5′-TCTTCATTGTGCTGGGTGC-3′

Protein analysis by western-blot

After treatment with As₄S₄, As³⁺ or a combination, NB4 and primary APL cells were collected, washed twice with PBS, and then lysed in ice-cold RIPA cell lysis buffer (Beyotime, China) containing 1.0 mM PMSF for 60 min to extract total cellular protein [28,29]. The concentration of total protein was determined using the BCA protein quantification kit (Beyotime, China). 25.0 μg of total protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Millipore, USA).The membrane was then blocked with 5% skim milk at room temperature for 60 min and sequentially incubated with primary and secondary antibodies. Proteins on the PVDF membrane were visualized using chemiluminescent HRP substrate (Millipore, USA). The band intensities were corrected using the β-actin intensities. All experiments were repeated at least three times.

Analysis of cell differentiation

We analyzed NB4 and primary APL cell differentiation with an FITC anti-human CD11b antibody using flow cytometry [19]. After treatment with As₄S₄, As³⁺, or a combination for 96 h, cells were harvested, washed twice with PBS and counted. A total of 1.0×10⁶ cells in 100 μl PBS were incubated with 20 μl FITC anti-human CD11b antibody at 4°C for 30 min. Excess antibody was washed out, and the percentages of FITC-CD11b-positive cells were analyzed using FlowJo.7.6.

Statistical analysis

Two-tailed Student’s t-tests were performed for comparisons of two groups, and P<0.05 was considered to be statistically significant.

Results

As₄S₄ enhances As³⁺-inhibition of NB4 and primary APL cell growth

The viability of NB4 and primary APL cells was determined by trypan blue exclusion [28]. Following 48 h of treatment, 97.5% of NB4 cells and 97.0% of primary APL cells were viable. The effects of As₄S₄, As³⁺, or their combination on NB4 and primary APL cell proliferation were analyzed using the WST-1 cell proliferation assay. After 48 h of treatment, As³⁺ and As₄S₄ obviously inhibited the growth of NB4 and primary APL cells (Fig 1A and 1B). The inhibitory potency of As³⁺ and As₄S₄ on cell growth increased with the increasing of concentration from 0.5 μM to 2.0 μM (Fig 1A and 1B). Subsequently, we investigated the combination effects of As³⁺ (0.5–1.5 μM) and As₄S₄ (0.5–1.5 μM) at fixed dose ratio (As₄S₄/As³⁺ were 1/3, 2/3, 1/1, 3/2, and 3/1) on the growth of NB4 and primary APL cells (Fig 1C). In our tested range of dose ratios, combining As₄S₄ and As³⁺ obviously inhibited the growth of NB4 and primary APL cells (Fig 1C). Compared with 2.0 μM As³⁺ alone or 2.0 μM As₄S₄ alone treated group, the combination of 1.0 μM As³⁺ and 1.0 μM As₄S₄ markedly induced the death of NB4 and primary APL cells (Fig 1D). In order to clarify whether As³⁺ and As₄S₄ exerted synergistic effects on cell growth, we analyzed CI values of the two drugs at different dose ratios (Fig 1E and 1F). When the dose ratio of As₄S₄/As³⁺ was in the range of 1:3 to 3:1, the combination of As³⁺ and As₄S₄ yielded a moderate synergistic effect (0.6≤CI<0.8) on the growth of NB4 and primary APL cells (Fig 1E and 1F) [30].

Fig 1. The effects of combining As³⁺ and As₄S₄ on the growth of NB4 and primary APL cells.

(A) The effects of As³⁺ on cell viability. (B) The effects of As₄S₄ on cell viability. (C&D) The combined effects of As³⁺ and As₄S₄ on cell viability. (E) CI of concurrent treatment with As³⁺ and As₄S₄ in NB4 cells. (F) CI in primary APL cells. CI<1.0 indicated a synergistic effect. The viability of NB4 and primary APL cells were determined by WST-1 cell proliferation assay kit after 48 h of treatment. Error bars represent the S.D. from the mean of three separate experiments. **P<0.01 compared with the control. ^##P<0.01 compared with As³⁺ and As₄S₄ combination treated cells.

As₄S₄ acts synergistically with As³⁺ to promote the apoptosis of NB4 and primary APL cells

Cell death comprises two major pathways: apoptosis and necrosis [20]. We investigated the effects of As₄S₄ on As³⁺-induced apoptosis in NB4 and primary APL cells by flow cytometry. As shown in Fig 2, Q₂ and Q₄ represent the percentages of Annexin V-FITC⁽⁺⁾/PI⁽⁺⁾ and Annexin V-FITC⁽⁺⁾/PI^(-) cells, respectively. After 48 h of treatment, both As³⁺ and As₄S₄ obviously induced NB4 cell apoptosis (Fig 2A). Compared with the control, the percentage of apoptotic cells in the group treated with 2.0 μM As₄S₄ increased from 3.7±1.3% to 19.4±2.6%, and the percentage reached 16.3±2.0% in the 2.0 μM As³⁺-treated group. Subsequently, we investigated the effects of combining 1.0 μM As₄S₄ and 1.0 μM As³⁺ on NB4 cell apoptosis. Compared with the groups treated with either 2.0 μM As₄S₄ or 2.0 μM As³⁺, the combination of 1.0 μM As₄S₄ and 1.0 μM As³⁺ markedly increased the proportion of apoptotic cells to 29.9±1.9% (Fig 2A and 2C). Similarly, As₄S₄ acted synergistically effects with As³⁺ on primary APL cell apoptosis (Fig 2B). Compared with the control, 2.0 μM As₄S₄ increased the percentage of apoptotic cells from 4.8±1.6% to 17.6±0.7%, and 2.0 μM As³⁺ increased the percentage of apoptotic cells to 16.8±2.6%. Furthermore, 1.0 μM As₄S₄ and 1.0 μM As³⁺ acted synergistically to increase the percentage of apoptotic cells to 28.0±1.5% (Fig 2B and 2C). The combination of As₄S₄ and As³⁺ synergistically promoted NB4 and primary APL cell apoptosis.

Fig 2. The effects of combining As³⁺ and As₄S₄ on the apoptosis of NB4 and primary APL cells.

(A) The apoptosis of NB4 cells. (B) The apoptosis of primary APL cells. (C) The percentage of apoptotic cells in NB4 and primary APL cells. After 48 h of treatment, the cells were stained with Annexin V-FITC and PI. Q₁ and Q_3, represent the dead cells and living cells, respectively. Q₂ and Q₄ were used to calculate the proportion of apoptotic cells. Figures show a representative experiment of three independent experiments. *P<0.05 and **P<0.01 compared with As³⁺ and As₄S₄ combination treated cells.

As₄S₄ and As³⁺ act synergistically to arrest the cell cycle in G₀/G₁ phase

The cell cycle is a highly regulated process, and aberrations in cell cycle distribution can induce abnormal cell changes such as apoptosis and differentiation [33,34]. The SubG₁ content represents the percentage of apoptotic cells [35]. In agreement with the results of the Annexin V-FITC and PI staining, As³⁺ and As₄S₄ obviously increased the SubG₁ contents (Fig 3). Furthermore, As₄S₄ enhanced As³⁺-induced apoptosis in NB4 and primary APL cells (Fig 3A and 3B). Compared with the groups treated with either 2.0 μM As³⁺ or 2.0 μM As₄S₄, combined treatment with 1.0 μM As³⁺ and 1.0 μM As₄S₄ for 48 h resulted in the increasing of SubG₁ content (Fig 3).

Fig 3. The effects of combining As³⁺ and As₄S₄ on cell cycle distribution.

(A) Cell cycle distribution in NB4 cells. (B) Cell cycle distribution in primary APL cells. (C) The percentage of cell cycle distribution in each phase. After 48 h of treatment, NB4 and primary APL cells were stained with PI and analyzed by flow cytometry. Figures show a representative experiment of three independent experiments. *P<0.05 and **P<0.01 compared with As³⁺ and As₄S₄ combination treated cells.

As³⁺ inhibited the G₁/S and S/G₂ transitions of NB4 and primary APL cells (Fig 3). In NB4 cells, As³⁺ increased the percentage of DNA at G₀/G₁ phase from 45.9±1.85% to 61.5±1.9%, and the percentage at G₂/M phase decreased from 7.1±0.9% to 2.3±0.6% (Fig 3A and 3C). In primary APL cells, As³⁺ increased the percentage of DNA at G₀/G₁ phase from 45.9±1.85% to 61.9±1.8%, and the percentage at G₂/M phase decreased from 7.1±0.9% to 2.1±0.8% (Fig 3B and 3C). As₄S₄, acting similarly to As³⁺, increased the percentage of DNA at G₀/G₁ phase and decreased the percentage at G₂/M phase (Fig 3). Compared with 2.0 μM As³⁺ alone or 2.0 μM As₄S₄ alone treated group, the combination of 1.0 μM As₄S₄ and 1.0 μM As³⁺ obviously blocked the G₁/S transition, as the DNA content at G₀/G₁ phase respectively reached 67.0±2.1% and 65.9±2.0% in NB4 and primary APL cells (Fig 3).

As₄S₄ has no obvious effects on As³⁺-induced ROS accumulation

As³⁺-induced apoptosis is related to cellular ROS accumulation [7,24]. To investigate whether As₄S₄-induced apoptosis in NB4 and primary APL cells is related to ROS generation, we detected cellular ROS with DCFH-DA fluorescence probe by flow cytometry. The results suggested that As₄S₄ obviously increased the percentage of DCF-positive cells, indicating an increase in cellular ROS levels (Fig 4A and 4B) [32]. Compared with the 2.0 μM As³⁺- or 2.0 μM As₄S₄-treated groups, the combination of 1.0 μM As³⁺ and 1.0 μM As₄S₄ did not obviously increase the percentage of DCF-positive cells (Fig 4A and 4B). We also analyzed the expression of HMOX1, a key oxidative stress response enzyme, to examine the effects of combining As³⁺ and As₄S₄ on cellular ROS [36]. RT-PCR analysis showed that 2.0 μM As³⁺ and 2.0 μM As₄S₄ up-regulated HMOX1 expression (Fig 4C). However, HMOX1 expression was not significantly different between cells treated with 2.0 μM As³⁺ and cells treated with both 1.0 μM As³⁺ and 1.0 μM As₄S₄ (Fig 4C and 4D). Notably, As₄S₄ induced ROS accumulation in NB4 and primary APL cells but did not affect As³⁺-induced ROS accumulation.

Fig 4. The effects of combining As³⁺ and As₄S₄ on cellular ROS accumulation.

(A) Cellular ROS were determined with DCFH-DA fluorescence probe in NB4 cells. (B) Cellular ROS in primary APL cells. (C) The effects of As₄S₄ and As³⁺ on HMOX1 expression. (D) The percentage of relative HMOX1 intensity obtained by RT-PCR. Error bars represent the S.D. from the mean of three independent experiments. *P<0.05 compared with As³⁺ and As₄S₄ combination treated cells.

As₄S₄ and As³⁺ regulate apoptotic factor expression

Bax, Bcl-2, caspase-3, NFκB, p53 and caspase-12 are the six factors that play key roles in As³⁺-induced cell apoptosis [21–25]. To clarify the mechanism of apoptosis, we investigated the effects of combining As₄S₄ and As³⁺ on the expression of these factors. RT-PCR and western-blot analysis suggested that both As₄S₄ and As³⁺ up-regulated the expression of pro-apoptotic factor Bax, down-regulated the expression of anti-apoptotic factor Bcl-2, and induced the activation of caspase-3 (Fig 5A and 5B). Compared with 2.0 μM As³⁺ alone or 2.0 μM As₄S₄ alone treated group, the combination of 1.0 μM As₄S₄ and 1.0 μM As³⁺ enhanced caspase-3 activation. However, their combined effects on Bax and Bcl-2 expression were not obvious (Fig 5C and 5D).

Fig 5. The effects of combining As₄S₄ and As³⁺ on mitochondria-mediated apoptosis.

(A) RT-PCR analysis of Bax, Bcl-2 and caspase-3 expression. (B) Western-blot analysis of Bax, Bcl-2 and caspase-3 expression. (C) Relative intensity of expression obtained by RT-PCR. (D) Relative intensity expression obtained by western-blot. Error bars represent the S.D. from the mean of three separate experiments. *P<0.05 and **P<0.01 compared with As³⁺ and As₄S₄ combination treated cells.

Subsequently, we investigated the effects of As₄S₄ and As³⁺ on NFκB, caspase-12 and p53 expression (Fig 6). As₄S₄ inhibited NFκB expression similarly to As³⁺ (Fig 6A and 6B). Moreover, As₄S₄ induced caspase-12 activation and promoted p53 tumor suppressor expression (Fig 6C and 6D). Compared with 2.0 μM As³⁺ alone or 2.0 μM As₄S₄ alone treated group, the addition of 1.0 μM As₄S₄ obviously promoted the regulation of 1.0 μM As³⁺ on NFκB, caspase-12 and p53 (Fig 6).

Fig 6. The effects of combining As₄S₄ and As³⁺ on NFκB, caspase-12 and p53 expression.

(A) RT-PCR and Western-blot analysis of NFκB expression. (B) Relative NFκB intensity obtained by RT-PCR and western-blot. (C) The effects of As₄S₄ and As³⁺ on caspase-12 and p53 expression. (D) Relative intensities of caspase-12 and p53 obtained by western-blot. Error bars represent the S.D. from the mean of three separate experiments. *P<0.05 and **P<0.01 compared with As³⁺ and As₄S₄ combination treated cells.

Low concentrations of As₄S₄ promote As³⁺-induced cell differentiation

Previous research suggested that As₄S₄ could induce NB4 cell differentiation [19]. However, the effects of As₄S₄ on As³⁺-induced NB4 cell differentiation have not been clarified. Herein, we investigated the effects of combining As₄S₄ and As³⁺ on NB4 and primary APL cell differentiation. As shown in Fig 7A, 0.1–0.4 μM As₄S₄ induced NB4 cell differentiation. When the concentration of As₄S₄ was greater than 0.3 μM, the percentage of CD11b-positive cells did not increase with the increasing of As₄S₄ concentration any more (Fig 7A). Compared with 0.4 μM As³⁺ alone- or 0.4 μM As₄S₄ alone-treated groups, the combination of 0.2 μM As₄S₄ and 0.2 μM As³⁺ obviously promoted NB4 cell differentiation (Fig 7B). Similarly, 0.1–0.4 μM As₄S₄ induced primary APL cell differentiation. Compared with the 0.4 μM As³⁺ and 0.4 μM As₄S₄ treatments, the percentage of CD11b-positive cells was obviously increased in the group treated with both 0.2 μM As₄S₄ and 0.2 μM As³⁺; however, the percentage of CD11b-positive cells was not increased in the group treated with 0.4 μM As₄S₄ and 0.4 μM As³⁺ (Fig 7C and 7D). Further investigation suggested that 0.1–0.25 μM As³⁺ and 0.1–0.25 μM As₄S₄ synergistically promoted the differentiation of NB4 and primary APL cells (data not show). The formation of the PML-RARα oncoprotein blocks NB4 and primary APL cell differentiation [8]. Therefore, we studied the effects of combining As₄S₄ and As³⁺ on PML-RARα expression by western-blot. After 96 h of treatment, 0.2 and 0.4 μM As₄S₄ induced PML-RARα oncoprotein degradation. Furthermore, As₄S₄ enhanced the As³⁺-induced degradation of the PML-RARα fusion protein in NB4 and primary APL cells (Fig 7E).

Fig 7. As₄S₄ acts synergistically with As³⁺ to affect NB4 and primary APL cell differentiation.

(A) The effects of As₄S₄ on CD11b expression in NB4 cells. (B) The effects of As₄S₄ on As³⁺-induced differentiation in NB4 cells. (C) The effects of As₄S₄ on As³⁺-induced differentiation in primary APL cells. (D) The percentage of FITC-CD11b-positive primary APL cells. (E) Western-blot analysis of PML-RARα expression in NB4 and primary APL cells. The figures show a representative experiment of three independent experiments. **P<0.01 compared with 0.2 μM As³⁺ and 0.2 μM As₄S₄ combination treated cells.

Discussion

As₄S₄, the major constituent of realgar, is another arsenic drug that has been used in traditional Chinese medicine for many years [16]. Since As³⁺ has been successfully used in APL treatment, the therapeutic potency of As₄S₄ in APL treatment has also been investigated [18,19]. The toxicity of ATO to organs, especially the liver and kidney, causes pain to patients [9,10]. As₄S₄ has low toxicity and good therapeutic potential for APL treatment, but it could not replace As³⁺. Pastorek M et al. have reported that the combination of As₄S₄ nanoparticles and As³⁺ induced dose-dependent activation of autophagy and apoptosis in melanoma cell lines [37]. Thus, Low concentrations of As³⁺ (0.1–1.0 μM) in combination with As₄S₄ (0.1–1.0 μM) may enhance the therapeutic efficacy of As³⁺ and reduce its adverse effects in the treatment of APL. Herein, we found that As₄S₄ and As³⁺ induced the apoptosis and differentiation of NB4 and primary APL cells in dose-dependent manner: 0.5–1.0 μM As³⁺ and 0.5–1.0 μM As₄S₄ synergistically promoted cell apoptosis; 0.1–0.25 μM As³⁺ and 0.1–0.25 μM As₄S₄ exerted synergistic effects on induction of cell differentiation.

To investigate the mechanism of apoptosis, we examined the effects of combining As₄S₄ and As³⁺ on the accumulation of cellular ROS and the expression of Bcl-2, Bax, NFκB, p53, caspase-12 and caspase-3. Mitochondria- and endoplasmic reticulum stress-mediated apoptosis pathways are always triggered by the accumulation of ROS [38,39]. Bcl-2 family members are key in mitochondria-mediated intrinsic apoptosis and caspase-3 activation [38]. The other organelle-controlled apoptosis pathway, endoplasmic reticulum stress-mediated apoptosis, is caused by multiple stimulations [39]. In response to stress, the endoplasmic reticulum induces the activation of caspase-12 and caspase-3, and promotes apoptosis [39]. As₄S₄ and As³⁺, the two different forms of arsenic, induced the accumulation of ROS, up-regulated the expression of the pro-apoptotic factor Bax, down-regulated the expression of the anti-apoptotic factor Bcl-2, and induced the activation of caspase-12 and caspase-3. Moreover, the synergistic effect of As₄S₄ on As³⁺-induced activation of caspase-12 and caspase-3 was obvious. However, the effects of combining As₄S₄ and As³⁺ on cellular ROS accumulation and Bcl-2 and Bax expression were not obvious. In addition to ROS, the p53 tumor suppressor also contributes to mitochondria-mediated and endoplasmic reticulum stress-mediated apoptosis [40,41]. Additionally, the p53 tumor suppressor regulates the G₁/S transition [24]. As³⁺ and As₄S₄ acted synergistically to arrest the cell cycle at G₀/G₁ and up-regulate p53 expression. Therefore, both ROS and p53 contributed to the apoptosis of NB4 and primary APL cells induced by As³⁺ and As₄S₄. We also studied the effects of combining As³⁺ and As₄S₄ on the extrinsic apoptosis pathway [23]. The results of RT-PCR and western-blot suggested that As₄S₄ and As³⁺ down-regulated NFκB expression, indicating that As₄S₄ and As³⁺ may promote NB4 cell apoptosis by inhibiting NFκB activation [23]. Above all, As₄S₄ and As³⁺ showed similar characteristics towards NB4 and primary APL cell apoptosis. Multiple pathways contributed to As₄S₄- and As³⁺-induced apoptosis. The combination of As₄S₄ and As³⁺ obviously promoted apoptosis by enhancing p53 expression, enhancing the endoplasmic reticulum stress-mediated pathway and inhibiting the NFκB signaling pathway.

Myeloid cell differentiation is regulated by many factors such as the CCAAT/enhancer-binding proteins, PU.1, and c-Myc [19]. NB4 and primary APL cells express the PML-RARα oncoprotein that prevents differentiation via the retinoic acid signaling pathway [19,42]. Moreover, As³⁺ was shown to control the fate of the PML-RARα oncoprotein through direct binding to the PML RING domain [8]. Therefore, we analyzed the effects of combining As₄S₄ and As³⁺ on the degradation of the PML-RARα oncoprotein in NB4 and primary APL cells. At low concentrations (0.1–0.4 μM), As₄S₄ promoted cell differentiation. The combination of 0.2 μM As³⁺ and 0.2 μM As₄S₄ obviously enhanced degradation of the PML-RARα oncoprotein and promoted differentiation of NB4 and primary APL cells via the retinoic acid signaling pathway.

Conclusions

As₄S₄ acts synergistically with As³⁺ towards NB4 and primary APL cell apoptosis and differentiation (Fig 8). As₄S₄ and As³⁺ induced the accumulation of cellular ROS and up-regulated the expression of the p53 tumor suppressor. ROS and p53 promoted mitochondria- and endoplasmic reticulum stress-mediated apoptosis by regulating Bcl-2 and Bax expression and inducing activation of caspase-12 and caspase-3. Concomitantly, As₄S₄ and As³⁺ synergistically inhibited NFκB activation to promote apoptosis. Moreover, low concentrations of As₄S₄ interacted synergistically with As³⁺ to induce degradation of the PML-RARα oncoprotein and promote NB4 and primary APL cell differentiation via the retinoic acid signal pathway. In this work, we found that the combination of As₄S₄ and As³⁺ could act synergistically to promote NB4 and primary APL cell apoptosis and differentiation, which may be a better therapeutic avenue for APL treatment.

Fig 8. Mechanism for the synergistic effects of As₄S₄ and As³⁺ on apoptosis and differentiation of acute promyelocytic leukemia cells.

Data Availability

All relevant data are within the paper.

Funding Statement

ZW was supported by the National Basic Research Program of China (2013CB922102), (http://www.973.gov.cn/English/Index.aspx) and the National Natural Science Foundation of China (21275072 and 21475059) (www.nsfc.gov.cn). ZG was supported by the National Natural Science Foundation of China (21201101) (www.nsfc.gov.cn). SW was supported by the Scientific Research Innovation Project for Postgraduates of Jiansu Province (CXZZ12-0038) (http://www.ec.js.edu.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.