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Introduction

Acute promyelocytic leukemia (APL) is a form of acute myeloid leukemia, which has been identified as the M3 subtype. A unique chromosome translocation t(15; 17)(q22; q21) found in the majority of APL patients leads to the formation of the promyelocytic leukemia retinoic acid receptor α (PML-RARα) fusion gene (1). The fusion protein encoded by the PML-RARα gene polymerizes and combines with retinoid-X receptor. The resultant protein complexes enhance histone deacetylase, thus repressing the transcription of the gene and disrupting the retinoic acid signal pathway under physiological concentrations of retinoic acid (1). This change results in the excessive growth of malignant promyelocytes and an inhibition of granulocyte differentiation.

All-trans retinoic acid (ATRA) as a successful model of differentiation therapy has improved the curative effect and extended the survival time of patients with APL. Clinical data has shown that the application of ATRA combined with chemotherapy increases the clinical complete response rate to ≤95% and the 2-year event-free survival rate was 86% (2). However, fatal retinoic acid syndrome and ATRA resistance in the majority of patients ultimately leads to treatment failure. Additionally, 31% of patients administered ATRA combined with chemotherapy relapse within 4–5 years after complete remission (3). Therefore, it is essential to identify new drugs for APL patients.

Arsenic trioxide (As2O3) and tetra-arsenic tetra-sulfide (As4S4), as traditional medicines, have been used widely for the treatment of newly diagnosed and relapse APL. The side effects, such as fluid retention, skin rashes, leukocytosis, gastrointestinal discomfort, pulmonary infiltrates, neuropathy, prolongation of the corrected QT interval, liver function abnormality and sudden death, make it difficult for APL patients to accept As2O3 as a single agent for long-term treatment (4). Therefore, As2O3 should be incorporated into combination medications with ATRA or used as a salvage therapy for relapse APL patients.

As4S4, which exerts similar effectiveness and less toxicity, provides not only a better quality-of-life, but is also advantageous in cytogenetic remission and PML-RARα reversion for newly diagnosed and hematological relapse patients. Following a single application of As4S4, the leukemia-free survival rate (LFS) for 1 and 3 years reached 86.1 and 76.6%, respectively, among newly diagnosed APL patients, with a median follow-up time of 13.5 months. In addition, the LFS for 1 and 6 years was 96.7 and 87.4% for the hematological complete remission group, with a median follow-up of 23 months (5). A previous study suggested that the LFS of APL patients at 2 years treated with As4S4 is higher than for those treated with As2O3 (6). Therefore, oral As4S4 is not inferior to intravenous As2O3 as an effective treatment for APL and may be considered a routine treatment option for the appropriate patients. The exact molecular mechanism of the drug's action remains unclear and warrants further investigation. The aim of the present study was to characterize the toxicity and apoptosis induced by As4S4 in a specific human APL NB4-R1 cell line that exhibits resistance to ATRA.

Materials and methods

Cell culture and reagents

The NB4-R1 APL-derived cells from an RA-resistant promyelocytic cell line were generously supplied by Shanghai Second Medical College (Shanghai, China). The NB4-R1 cells were cultured in RPMI-1640 (Gibco-BRL, Carlsbad, CA, USA) medium supplemented with 10% heat-inactivated (at 56°C for 30 min) fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin, and maintained in a 5% CO2 humidified atmosphere at 37°C. The cells were grown at an optimal cell density between 5×105 and 1×106/ml. Cell viability was evaluated via trypan blue dye exclusion assays and the cell survival rate was >95%. As4S4 (Xi'an Traditional Chinese Drug Company, Xi'an, China) stock solution was obtained by dissolving in the 1.0 M NaOH assistant agent. According to the IC50 of NB4-R1 cells in our previous study (7), the exponentially growing cells were treated with 25 µmol/l As4S4 for 0, 24 or 48 h and the cells were analyzed via flow cytometry, DNA ladder electrophoresis and western blot analysis.

Measurement of apoptosis

Flow cytometric analysis using Annexin V-FLUOS and PI (Roche Custom Biotech, Indianapolis, IN, USA) was performed to differentiate between live, apoptotic and necrotic cells following treatment with As4S4. Subsequent to the treatment with 25 µmol/l As4S4 for 0, 24 or 48 h, 1×106 cultured cells were harvested and washed twice with cold phosphate-buffered saline (PBS). The cells were centrifuged at 200 × g for 5 min at 4°C and re-suspended in 100 µl of Annexin V-FLUOS/PI labeling solution for 10–15 min in the dark at room temperature. The stained cell suspension was immediately analyzed using a flow cytometer (BD Biosciences FACSCalibur double laser flow cytometer; BD Biosciences, Franklin Lakes, NJ, USA). The data analysis was performed using the CellQuest software program (BD Biosciences).

DNA ladder agarose gel electrophoresis

DNA ladder fragmentation reflecting the endonuclease activity is a characteristic feature of apoptosis. After incubation for 0, 24 or 48 h with As4S4, the NB4-R1 cells were collected and washed twice with PBS. Subsequently, 1×106 cells were solubilized and the chromosomal DNA was extracted and purified using an Apoptotic DNA Ladder kit (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer's instructions. The DNA samples were electrophoresed on a 1.5% agarose gel containing 1 mg/ml ethidium bromide at 60 V for 2 h. The apoptotic DNA fragments were analyzed and photographed using a Quantity One gel image analysis system (ChemiDOC XRS; Bio-Rad, Richmond, CA. USA).

Cell cycle analysis

The cell cycle distribution was analyzed via flow cytometry (BD Biosciences FACSCalibur double laser flow cytometer). Following treatment with As4S4 for 0, 24 or 48 h, the NB4-R1 cells (1×106) were harvested and washed twice with ice-cold PBS. The cells were suspended gently in 70% chilled ethanol at −20°C overnight. After washing with PBS, the cells were re-suspended in 500 µl PBS containing PI (50 µg/ml) and RNase (50 µg/ml), and were incubated for 30 min at room temperature in the dark. The cell cycle phase distribution of each experiment was analyzed using 10,000 cells per sample. The proportion of cells in the G0/G1, S and G2/M phases were represented as DNA histograms.

Western blot analysis

After treatment with 25 µmol/l As4S4 for 0, 24 or 48 h, the cultured cells were harvested and washed three times with cold PBS. Subsequently, the cells were solubilized in radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail (Sigma, St. Louis, MO, USA). After incubation on ice for 10 min, the cell suspension was centrifuged for protein at 15,500 × g for 15 min at 4°C. The protein (30 µg) was separated on 10% SDS-PAGE and transferred to a nitrocellulose membrane at 110 V for 2 h. The non-specific binding sites on the membranes were blocked with 5% (w/v) skimmed milk in Tris-buffered saline (TBS): [20 mmol/l Tris-HCl and 200 mmol/l NaCl (pH 7.6)] for 2 h under gentle agitation at room temperature. Subsequently, the membranes were incubated with the relevant primary antibodies [poly ADP-ribose polymerase (PARP) rabbit monoclonal, 1:10,000; Cell Signaling Technology, Inc., Danvers, MA, USA; B-cell lymphoma 2 (Bcl-2) mouse monoclonal, 1:1,000; Bax rabbit monoclonal, 1:1,000; caspase-3 rabbit monoclonal, 1:1,000; GAPDH mouse monoclonal, 1:10,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA] directed against the protein or enzyme of interest for 1 h at room temperature and subsequently at 4°C overnight. The membranes were washed extensively with TBS containing 0.05% Tween-20 (v/v) (TBST) and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Following washing with TBST, the membranes were incubated under chemiluminescence and wrapped in clear plastic wrap for film exposure. The bands on the immunoblots were quantified using Quantity One version 4.6.2 software (Bio-Rad). The protein expression of each sample was internally normalized to GAPDH and the quantity was compared with the expression of the control groups.

Statistical analysis

Experiments were performed in duplicates or triplicates of ≥3 independent experiments and the results are presented as the mean ± standard deviation. Statistical analysis between groups was carried out via a one-way analysis of variance using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

As4S4 induces NB4-R1 cell apoptosis in a time-dependent manner

Apoptotic characterization was performed in the As4S4-treated cells via Annexin V-FLUOS and PI double staining and the samples were analyzed via flow cytometry. The data revealed that the untreated cells showed normal cell viability. In contrast to the control cells, the percentage of early apoptotic cells treated with 25 µmol/l As4S4 for 24 or 48 h (Annexin V+/PI−, in the lower right quadrant) significantly increased from 0.00 to 24.49 and 47.41% (P<0.05), respectively. In addition, the percentage of late apoptotic cells (Annexin V+/PI+, shown in the upper right quadrant) significantly increased from 0.08 to 14.72 and 20.70% (P<0.05), respectively. As shown, a progressive increase in the number of apoptotic cells was observed, which suggests time-dependent cytotoxicity (Fig. 1, Table I).

Figure 1. Apoptosis of the NB4-R1 cells treated with tetra-arsenic tetra-sulfide (As4S4) via flow cytometry. (A) Control; (B) As4S4 treatment for 24 h; and (C) As4S4 treatment for 48 h. PI, propidium iodide.

Table I. Flow cytometric data showing the effect of As4S4 (25 µmol/l) on Annexin V/PI binding in the NB4-R1 cells.

Table I.

Flow cytometric data showing the effect of As4S4 (25 µmol/l) on Annexin V/PI binding in the NB4-R1 cells.

Action time, h	Viable cells	Early apoptotic cells	Late apoptotic cells
0	99.53±0.39	0.00±0.00	0.08±0.13
24	60.27±5.12a	24.49±4.05a	14.72±1.82a
48	31.60±1.48a	47.41±4.78a	20.70±3.89a

a P<0.05, significant difference compared to the control group. Data are mean % ± SD; n=3. As₄S₄, tetra-arsenic tetra-sulfide; PI, propidium iodide; SD, standard deviation.

DNA ladder agarose gel electrophoresis was used to distinguish apoptotic cells from necrosis. Apoptosis is characterized by internucleosomal DNA ladder fragmentation through agarose gel electrophoresis to show a ‘ladder’ pattern at 180-base pair (bp) intervals due to the activation of endogenous endonucleases, whereas random DNA fragmentation is a typical manifestation of necrosis following electrophoretic separation. The untreated cells contained only high-molecular-weight genomic DNA. Compared with the control group, the NB4-R1 cells inoculated with As4S4 exhibited the characteristic pattern of nucleosomal laddering specific to apoptosis, which was visible as faint bands on the gel. As4S4 produced DNA fragments of low molecular weight consisting of multimers of 180–200 bp in the NB4-R1 cell line in the 24-h treatment groups. In the 48-h treatment groups, DNA degradation failed to form the typical bands but formed random DNA fragmentation, which indicates necrosis of cells at this time point (Fig. 2).

Figure 2. Apoptosis in the NB4-R1 cells treated with tetra-arsenic tetra-sulfide (As4S4) assessed by analyzing the DNA ladder fragmentation. The NB4-R1 cells were treated for 0, 24 or 48 h. M, DNA marker; bp, base pairs.

Effect of As4S4 on the proteins associated with NB4-R1 cell apoptosis

Based on the above result, the association between apoptosis factor expression and apoptosis induction was investigated in the NB4-R1 cells treated with As4S4. The expression of several apoptosis-related factors was studied. Bax levels increased significantly over the control, whereas Bcl-2 showed a clear reduction. All the factors exhibited variations in a time-dependent manner.

As changes of Bax/Bcl-2 have been reported to play significant roles in the activation of caspase signaling, the activation of caspase-3 was detected in the following set of experiments. Incubation of the NB4-R1 cells with As4S4 for 24 h induced activation of caspase-3. Pro-caspase-3 was cleaved into small active fragments of 19 or 17 kDa under apoptotic stimulation. The 113 kDa PARP, as the specific substrate of caspase-3, was cleaved into 89 and 24 kDa fragments after treatment for 24 h. No cleavage of PARP in the control group was detected (Fig. 3).

Figure 3. Expression of cleaved poly ADP-ribose polymerase (PARP), B-cell lymphoma 2 (Bcl-2), Bax and cleaved caspase-3 via western blot analysis. The NB4-R1 cells were treated with tetra-arsenic tetra-sulfide (As4S4) for 0, 24 or 48 h.

As4S4 induces cell cycle arrest in NB4-R1 cells

Flow cytometry assays were performed to analyze the effects of As4S4 on the cell cycle distribution. According to the DNA histogram results, the drug induced a significant increase in the cell population in the S phase. Cells in the S phase increased from 31.85% of untreated cells to 42.53 and 55.12% of cells in the experimental groups (P<0.05), where the action of As4S4 induced nearly a 2-fold increase. Compared to the control group, the distribution of NB4-R1 cells in the G0/G1 phases decreased from 57.29 to 37.57 and 28.51% (P<0.05), respectively. The percentage of G2/M phase cells detected at different time points increased from 10.79 to 19.91 and 17.01%, representing a slight increase compared to the controls (P<0.05). These results show that the inhibitory effect on the growth of NB4-R1 cells induced by As4S4 was partially mediated by reducing the number of cells in the G0/G1 phases and arresting the cell cycle in the S phase and G2/M phases (Table II, Fig. 4).

Figure 4. Influence of tetra-arsenic tetra-sulfide on the cell cycle of NB4-R1 cells.

Table II. Flow cytometric data showing the effect of As4S4 (25 µmol/l) on the progression of the cell cycle in NB4-R1 cells.

Table II.

Flow cytometric data showing the effect of As4S4 (25 µmol/l) on the progression of the cell cycle in NB4-R1 cells.

Action time, h	G0/G1	S	G2/M
0	57.30±0.35	31.85±0.91	10.79±0.67
24	37.56±1.85a	42.53±2.71a	19.91±1.83a
48	28.51±2.53a	55.12±0.13a	17.01±1.44a

{ label (or @symbol) needed for fn[@id='tfn2-br-0-0-466'] } Data are mean % ± SD. n=3.

a P<0.05, significant difference compared to the control group. As₄S₄, tetra-arsenic tetra-sulfide; SD, standard deviation.

Discussion

APL accounts for 10–15% of acute myeloid leukemia in adults (1). As4S4 has gained importance in the treatment of the APL. Previous studies have shown that the therapeutic action of As4S4 is also effective for other tumor therapies (8,9). However, the molecular mechanism of the action of As4S4 in RA-resistant APL therapy remains unknown. The present results reveal a time-dependent toxic action of As4S4 on RA-resistant NB4-R1 cells. Flow cytometric analyses and DNA ladder agarose gel electrophoresis confirmed that As4S4 inhibited tumor cell growth via inducing apoptosis. To probe the cell signaling pathways involved in this As4S4-induced apoptosis, the protein expression levels of Bcl-2, Bax, caspase-3 and PARP were detected via western blot analysis.

The Bcl-2 family of pro- and anti-apoptotic proteins plays an important role in apoptosis that is induced by a variety of stimuli. Bcl-2 proteins modulate the integrity of the mitochondrial and endoplasmic reticulum membranes, cytochrome c release, caspase activation and cell death (10). A reduction in Bcl-2 expression can lead to a loss of signals that are required for survival. Bax is a major pro-apoptotic member that is required for apoptotic cell death. Previous evidence has indicated that Bcl-2 can constitute homodimers and heterodimers with Bax, leading to an inhibition of the formation of Bax/Bax pro-apoptotic homodimers (11,12). The ratio between anti-apoptotic and proapoptotic members of the Bcl-2 family may determine the susceptibility of the cell to apoptosis. The present study reported a decrease in Bcl-2 and an increase in Bax following treatment of the NB4-R1 cells with As4S4. The decrease in the Bcl-2/Bax ratio leads to the translocation of Bax from the cytoplasm to mitochondria, promoting the release of cytochrome c and the activation of caspase. Variations in the levels of Bax and Bcl-2 can be deduced by apoptosis that is initiated via the intrinsic pathway.

Caspase-3, as the most important executor of apoptosis, participates in DNA degradation, nuclear condensation, plasma membrane blebbing and proteolysis of certain caspase substrates (13,14). Caspases are synthesized as relatively inactive precursors (zymogens) that require proteolytic processing for activation. As discovered in the NB4-R1 cells, As4S4 cleaves the 36-kDa pro-caspase-3 into small 17 or 19 kDa active fragments, leading to caspase-dependent apoptosis. Subsequently, the cleaved caspase-3 activates endonuclease caspase-activated DNase, leading to fragmentation of the chromosomal DNA at internucleosomal sites (15). The present results show that cleaved caspase-3 significantly increased after As4S4 incubation for 24 h while the DNA degradation revealed characteristic DNA ‘ladder’ bands. The activity of this endonuclease can be inhibited by PARP and the cleavage of PARP by activated caspase-3 reverses the activity of the endonuclease (16). In the present study, the 113-kDa PARP could be cleaved onto an 89-kDa C-terminal catalytic fragment and an N-terminal 24-kDa fragment after 24 h of As4S4 treatment, leading to a loss of DNA repair function.

Various chemotherapy drugs inhibit the growth of tumor cells by blocking the cell cycle. Numerous investigators have reported that As4S4 blocks tumor cells at different stages of the cell cycle (17,18). Variations in experimental results may be associated with drug concentration, action time and cell types. In the present study, the accumulation of cells in the S and G2/M phases was observed for NB4-R1 cells, suggesting that As4S4 may exert its cytotoxic effects on NB4-R1 cells through cell cycle arrest and cell apoptosis.

In conclusion, the present study revealed that As4S4, a traditional medicine, inhibited the growth of NB4-R1 cells in vitro. As4S4 induced cell apoptosis through changes in Bcl-2 and Bax, activation of caspase-3 and cleavage of PARP. The results suggested the apoptosis of NB4-R1 cells via a mitochondria-dependent pathway. In addition, As4S4 may exert its cytotoxic effects on NB4-R1 cells through blocking the cell cycle in the S and G2/M phases. Thus, As4S4 may be a potential anticancer drug candidate. The development of cell apoptosis is a multi-factor, multi-step and multi-gene interactive process. The signaling pathways and molecular mechanisms of As4S4 in apoptotic regulation require further investigations.

Acknowledgements

The present study was supported by the Natural Science Foundation of China (grant no. 81000218). The authors would like to express their gratitude to Dr Xinyang Wang for access to the Oncology Research Laboratory, Key Laboratory of Environment and Genes Related to Diseases (Xi'an, China) to complete the experiments.

References