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Introduction

Arsenic is a widespread environmental metalloid present in nature as oxide and sulfide compounds. Inorganic arsenic compounds comprise numerous valence states, including arsenic trioxide (As2O3), realgar (As2S2), As (III), and As (V). As (III) has been used treat tumors with a positive effect; although, due to its' higher solubility and bioavailability, it may be more toxic than As (V) (1). Organoarsenic compounds comprise liver detoxification metabolites, including dimethylarsenic acid [DMA; (CH3)2AsO2H] and 4-[N-(S-glutathionylacetyl) amino] phenylarsonic acid (GSAO) (2). It has been demonstrated that As2O3 treats acute promyelocytic leukemia due to its antitumorigenic properties (3,4). Furthermore, in vitro studies have revealed that As2O3 induces apoptosis in various types of cell line, including the DU145 and PC-3 (prostate cancer) (5), MDAH 2774 (ovarian cancer) (5) and TM4 (sertoli tumor) cell lines (6), and CD133+/CD13+ liver cancer stem cells (7). In addition, GSAO, an organoarsenic compound, has been reported to inhibit proliferation in endothelial and tumor cells, such as fibrosarcoma cells, lung cancer, pancreatic cancer and prostate cancer cells in vitro, and reduce angiogenesis and tumor growth in a xenograft mouse model (8,9). Furthermore, DMA exerts an antiproliferative and cytotoxic effect on human leukemia and multiple myeloma cells (10). Inorganic and organic arsenic compounds therefore represent novel potential therapeutic agents against solid tumors and various types of malignancy (3–10).

Previous studies have reported that tumor formation arises from an imbalance between cell proliferation and apoptosis. Subsequently, apoptosis induction is a potential target for cancer therapies (11,12). Apoptosis is induced by various types of caspase, which are aspartatespecific cysteine proteases, during the extrinsic and intrinsic apoptosis pathways that involve death receptors and mitochondria, respectively. The extrinsic pathway is initiated from death receptors, e.g., tumor necrosis factor (TNF) receptor and Fas, which bind with their associated ligands, i.e., TNF and Fasligand (FasL), prior to subsequent caspase-8 activation (13). The intrinsic pathway is caused by mitochondrial dysfunction, which leads to cytochrome c release in the cytosol and subsequent formation of the apoptosome when combined with cleaved caspase-9 (14). These two caspase cascades eventually trigger caspase-3 activation and subsequent cellular morphological alterations, including membrane blebbing, phosphatidylserine externalization, cell detachment and chromosomal DNA fragmentation (15). In addition, proteins from the B-cell lymphoma-2 (Bcl-2) family are key regulators of the apoptotic response. They serve different physiological roles in mitochondrial integrity, including multidomain antiapoptotic (e.g. Bcl-2 and Bcl-extra-large), multidomain proapoptotic (e.g. Bcl-2 associated X, apoptosis regulator and Bcl-2 antagonist/killer), and Bcl-2 homology region 3 (BH3)-only proapoptotic (e.g. BH3 interacting domain death agonist and Bcl-2 modifying factor) roles (16). These proteins can positively and negatively regulate mitochondrial permeability and apoptotic protein efflux (17–19). A previous study demonstrated that As2O3 upregulates BH3-only proapoptotic, and downregulates antiapoptotic, protein levels in myeloma (20). In addition, the extrinsic apoptotic pathway, which involves Fas/FasL, also participates in arsenic-induced keratinocyte apoptosis (21). The mechanisms underlying arsenic-induced apoptosis in various types of tumor cell are therefore complex, and have yet to be fully elucidated.

Leydig cell tumors are one type of sex cord-stromal malignancy observed in testicular cancer, accounting for 1–3% of all testicular neoplasms and 4–9% of tumors of the testis in prepubertal boys. Epidemiological studies have reported that the incidence of testicular cancer has been increasing worldwide over the past 30 years (22). Clinically, the major therapeutic strategy for Leydig cell tumor is radical orchiectomy. Testis sparing surgery is preferred in order to maintain fertility. In addition, ~10% of Leydig cell tumors respond unfavorably to chemotherapy and irradiation (23). The present study aimed therefore to explore alternative therapeutic strategies to treat Leydig cell tumors. Particularly, this study aimed to determine the mechanisms underlying the arsenicinduced cell apoptosis in Leydig cell tumors. To do so, the effect of arsenic compounds, including sodium arsenite and DMA, which are the most representative inorganic and organic arsenite compounds, respectively (8), were investigated in MA-10 mouse Leydig tumor cells, which may aid the development of potentially more effective chemotherapy strategies.

Materials and methods

Chemicals

Sodium arsenite was purchased from Fluka (St. Gallen, Switzerland). DMA, RNase A, Waymouth's MB 752/1 medium, propidium iodide (PI), Folin & Ciocalteu's phenol reagent, EDTA, EGTA, 30% acrylamide/Bisacrylamide solution, MTT and anti-β-actin monoclonal antibody were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Fetal bovine serum (FBS) and trypsin-EDTA were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Gentamycin sulfate was purchased from AG Scientific Inc. (San Diego, CA, USA). Sodium chloride (NaCl), HEPES, potassium chloride and Tris base were purchased from J.T. Baker (Phillipsburg, NJ, USA). Disodium hydrogen phosphate, potassium dihydrogen phosphate, and tissue culture grade sodium bicarbonate were purchased from Riedel-de Haën (Seelze, Germany). Hydrochloric acid, sodium dodecyl sulfate (SDS), Tween-20 and dimethyl sulfoxide (DMSO) were purchased from Merck KGaA. Sucrose was purchased from Panreac (Barcelona, Spain). The general caspase inhibitor Z-VAD-fmk was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Goat horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin (Ig)G (cat. no. NEF812001EA; 1:2,000) and HRP-conjugated antimouse IgG secondary antibodies (cat. no. NEF822001EA; 1:2,000) were purchased from PerkinElmer, Inc. (Waltham, MA, USA). The Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was purchased from Strong Biotech (Taipei, Taiwan). Polyclonal antibodies against cleaved caspases-3 (cat. no. 9661; 1:2,000), cleaved caspase-8 (cat. no. 9429; 1:2,000) and cleaved caspase-9 (cat. no. 9509; 1:2,000) and poly(ADP-ribose) polymerase (PARP; cat. no. 9542; 1:2,000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The enhanced chemiluminescence (ECL) detection kit was purchased from EMD Millipore (Billerica, MA, USA).

Cell culture and treatments

The MA-10 cell line was kindly donated by Dr Mario Ascoli (University of Iowa, Iowa City, IA, USA), and maintained using standard techniques as previously described (24,25). Briefly, cells were cultured in Waymouth's MB 752/1 medium supplemented with 10% FBS and placed at 37°C in a humidified incubator containing 5% CO2. A total of 6×106 MA-10 cells were plated in a 60-mm dish. After 24 h, MA-10 cells were treated with 0.01–100 µM sodium arsenite, 0.1 µM-10 mM dimethylarsenic acid or PBS (Control) in the medium containing 1% FBS for 3, 6, 12 and 24 h.

Morphological observation

The morphology of arsenic compound-treated MA-10 cells and control cells at 3, 6, 12 and 24 h after treatment was examined using an Olympus CK40 light microscope (Olympus Corporation, Tokyo, Japan) and images were captured using an Olympus DP20 digital camera (Olympus Corporation; magnification, ×200). Apoptosis was characterized by assessing plasma membrane blebbing and detached cells, as described previously (26).

Cell viability assay

Cell viability was assessed with MTT assay as described previously (27), following treatment with arsenic compounds. In brief, MA-10 cells were seeded in 96-well plates at a density of 8×103 cells per well and 50 µl MTT (0.5 mg/ml) was added to each well after 24, 48 and 72 h after treatment with arsenic compounds, and incubated at 37°C for 4 h. The supernatant was discarded and the MTT-formazan crystals were dissolved with 50 µl 0.5% DMSO in each well for 1 h. The optical density (OD) values were read using a VersaMax ELISA microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) at a wavelength of 570 nm.

Cell cycle analysis

Cell cycle distribution was determined by flow cytometry following PI staining (28). After 18 h of serum starvation, MA-10 cells were treated with arsenic compounds for 3, 6, 12 and 24 h, and then cells were detached using 1% trypsin and fixed with 70% ethanol for 2 h at −20°C. Fixed cells were simultaneously treated with RNase (100 µg/ml) and PI (40 µg/ml) for 30 min at room temperature and analyzed using flow cytometry on a FACScan™ (Becton-Dickinson and Company, Franklin Lakes, NJ, USA) with an excitation wavelength of 488 nm and a band pass filter >600 nm. Cells in the sub-G1 phase, also termed hypodiploid, contain less DNA, which is due to apoptosisassociated DNA fragmentation (29).

Annexin V/PI double staining assay

A total of 6×106 MA-10 cells were seeded in a 60-mm dish and treated 1 day after seeding with 10 or 100 µM sodium arsenite, 1 or 10 mM dimethylarsenic acid or PBS in the medium containing 1% FBS for 24 h. Treated-MA-10 cells were collected using trypsin and centrifuged at 120 × g for 10 min at 4°C, and then incubated with 100 µl Annexin V-FITC staining solution (apoptosis detection kit; Strong Biotech Corporation, Taipei, Taiwan) for 15 min at room temperature according to the manufacturer's protocol. Samples were analyzed on a FACSCalibur flow cytometer (Becton-Dickinson and Company) with an excitation wavelength of 488 nm and band pass filters of 515 and 600 nm for FITC and PI detection, respectively. Data were represented using histogram plots gated into four quadrants containing negative (Annexin V/PI), PIpositive (Annexin V/PI+), Annexin Vpositive (Annexin V+/PI) and Annexin V/PIdoublepositive (Annexin V+/PI+) stained cells, which corresponded to viable, dead, early apoptotic and late apoptotic cells, respectively (30).

Protein extraction and western blotting

Treated cells were lysed with 100 µl ice-cold lysis buffer for 30 min at room temperature, which contained 20 mM Tris at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate and 1 mM sodium orthovanadate. Lysates were centrifuged at 12,000 × g for 12 min at 4°C, and supernatants were collected and stored at 20°C until future analysis. Protein concentration was determined using the Lowry protein assay as described previously (31). Proteins (30 µg) were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% milk dissolved in TBST for 1 h at room temperature, and incubated with primary antibodies overnight at 4°C. Membranes were then washed three times with TBST and incubated for 1 h with the appropriate HRP conjugated secondary antibodies. Bands were detected using ECL substrate and the UVP EC3 BioImaging system (UVP, LLC, Phoenix, AZ, USA) (32–34). Quantification of the western blotting data was performed using ImageJ version 1.50 software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

All data were expressed as the means ± standard error of the mean from three separate experiments. The statistical significance of the differences between the control and treatment groups at various time-points was determined using a two-way analysis of variance followed by the Least Significance Difference test. Statistical analysis was performed using GraphPad Prism version 6 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Effects of arsenic compounds on MA-10 cell morphology and viability

To determine the cytotoxicity of arsenic compounds, MA-10 cells were treated with or without sodium arsenite (0.01–100 µM) and DMA (0.1–10 mM) for 3, 6, 12 and 24 h. At each time point, untreated MA-10 cells were firmly attached and exhibited the commonly anticipated polygonal-shaped morphology (Fig. 1A). Conversely, cells treated with 100 µM sodium arsenite or 10 mM DMA became gradually rounded and more detached over time (Fig. 1A and B, respectively). However, 12 and 24 h of treatment with 10 µM sodium arsenite, and 24 h of treatment with 1 mM DMA, induced plasma membrane blebbing and an enlarged and flattened appearance, which suggested that cell apoptosis was occurring (Fig. 1A and B). Sodium arsenite appeared to be more toxic than DMA to MA-10 cancer cells. However, low concentrations of sodium arsenite (0.01, 0.1 and 1 µM) or DMA (0.1, 10 µM and 10 mM) did not effect cell morphology.

Figure 1. Effect of sodium arsenite and dimethylarsenic acid on cell morphology and viability. MA-10 cells were treated with sodium arsenite (0.01–100 µM) and dimethylarsenic acid (0.1 µM-10 mM) for 3, 6, 12 and 24 h. Morphological changes of MA-10 cells following (A) sodium arsenite and (B) dimethylarsenic acid treatments were observed and recorded under a light microscope. Magnification, ×200; scale bar, 50 µm). Arrowheads represent the membrane blebbings. MA-10 cell viability following (C) sodium arsenite and (D) dimethylarsenic acid treatments were quantified by MTT viability assay. Results are expressed as the percentages of cell growth relative to control (100%). Data in (C) and (D) represent the mean ± standard error of the mean of three separate experiments. *P<0.05.

The effects of arsenic compounds on MA10 cell morphology were further investigated with the MTT viability assay. MA10 cell viability significantly decreased to 88±2.9, 84±7.4, 74±3.8 and 45±6.4% following 3, 6, 12 or and 24 h treatment with 10 µM sodium arsenite, respectively, compared with the control group (Fig. 1C). MA-10 cell treatment with 10 mM DMA only significantly reduced cell viability to 99±6.9, 79±6.6, 84±0.8 and 49±6.0% at the same time points (Fig. 1D). These results indicated that sodium arsenite and DMA both induced time and dosedependent cell toxicity, and that sodium arsenite was more potent (given the 1,000-fold difference in the concentration of the two compounds that elicited these effects).

Arsenic compounds induce MA10 cell apoptosis

To determine whether MA10 cells were undergoing apoptosis following treatment with arsenic compounds, cell DNA content was quantified using PI staining. A significant increase in the number of cells in the sub-G1 phase, which is an apoptotic marker, was observed following 12 and 24 h treatment with 10 µM sodium arsenite or 10 mM DMA, respectively (P<0.05; Fig. 2A and C). In addition, the cell population in the G2/M phase was significantly increased following 24 h treatment with 10 and 100 µM sodium arsenite, and 12 h treatment with 1 mM DMA (12 and 24 h), which indicated that both arsenic compounds caused G2/M cell cycle arrest of MA10 cells (Fig. 2B and D) (P<0.05). These results suggested that cell cycle regulation and redistribution may be involved in sodium arsenite- and DMA-induced MA-10 cell apoptosis.

Figure 2. Effect of sodium arsenite and dimethylarsenic acid on cell cycle distribution. MA-10 cells were treated without or with sodium arsenite (0.01–100 µM) and dimethylarsenic acid (0.1 µM-10 mM) for 3, 6, 12 and 24 h. Cells were stained with PI and analyzed for cell cycle distribution by flow cytometry. (A) and (B) represent sub-G1 and G2/M phases, respectively, following MA-10 cell treatment with sodium arsenite. (C) and (D) represent sub-G1 and G2/M phases, respectively, following MA-10 cell treatment with dimethylarsenic acid. Data represent the means ± standard error of the mean of three separate experiments. *P<0.05.

Annexin V/PI double-staining was used to refine the apoptosis stages observed in MA-10 cells (Fig. 3A). A shift in alive (Annexin V−/PI−), dead (Annexin V−/PI+), early apoptotic (Annexin V+/PI−) and late apoptotic (Annexin V+/PI+) cell populations was observed following 24 h treatment with 10 and 100 µM sodium arsenite, and with 1 and 10 mM DMA (Fig. 3B and C, respectively). Notably, the differences in the Annexin V+-stained cells (early and late apoptotic status) were further analyzed following sodium arsenite (Fig. 3D) and DMA (Fig. 3E) treatments. The results demonstrated that both arsenic compounds significantly induced MA-10 cell apoptosis following 24 h, as indicated by the increased ratio of Annexin V+ cells (P<0.05). These results further suggested that sodium arsenite and DMA may be cytotoxic to Leydig cancer cells, particularly DMA.

Figure 3. Effect of sodium arsenite and dimethylarsenic acid on cell apoptosis. MA-10 cells were treated without or with sodium arsenite (10 and 100 µM) and dimethylarsenic acid (1 and 10 mM) for 12 and 24 h. (A) Apoptotic status of the arsenic-treated cells was determined by Annexin V/PI double staining. The proportion of negative (viable) cells, Annexin V-positive (early apoptotic) cells, PI-positive (dead) cells, and Annexin V and PI double-positive (late apoptotic) stained cells are shown following (B) sodium arsenite (10 and 100 µM) and (C) dimethylarsenic acid (1 and 10 mM) treatments. The differences in Annexin V-positive stained cells (early and late apoptotic) following (D) sodium arsenite and (E) dimethylarsenic acid treatments were further analyzed. Data represent the means ± standard error of the mean of three separate experiments. *P<0.05. FITC, fluorescein isothiocyanate; PI, propidium iodide.

Arsenic-induced MA-10 cell apoptosis is mediated by caspase cascade

The caspase cascades are essential regulators of the apoptotic signaling transduction pathway (12). Activation of these cascades triggers PARP cleavage of its 85 kDa C-terminal fragment. PARP cleavage, which is a common apoptosis marker, follows caspase-3 activation and cleavage (35). To investigate whether arsenic compounds affect the extrinsic and/or intrinsic apoptotic pathways, the protein levels of cleaved (activated) caspases-3, −8 and −9 were assessed by western blotting. The results demonstrated that 12 and 24 h treatment with 10 µM sodium arsenite significantly increased cleaved caspase-8 and −3 protein levels (P<0.05; Fig. 4A, B and D). In addition, 24 h treatment with 10 µM sodium arsenite significantly increased the cleaved caspase-9 protein level (P<0.05; Fig. 4A and C). Furthermore, 12 h treatment with 10 µM sodium arsenite significantly increased the cleaved PARP protein level (P<0.05; Fig. 4A and E). Treatment with 1 mM DMA for 24 h significantly increased the cleaved caspase-8 protein level (P<0.05; Fig. 5A and B). In addition, 24 h treatment with 1 and 10 mM DMA significantly elevated the cleaved caspase-9 protein level (P<0.05; Fig. 5A and C). Furthermore, 12 and 24 h treatment with 10 mM DMA significantly increased the cleaved caspase-3 protein level (P<0.05; Fig. 5A and D). Finally, treatment with 10 mM DMA for 12 h significantly increased the cleaved PARP protein level (P<0.05; Fig. 5A and E). These results demonstrated that arsenic compounds are involved in the intrinsic and extrinsic apoptotic pathways in MA-10 cancer cells. In addition, these results further indicated that sodium arsenite was more potent than DMA in terms of activating caspase pathways in MA10 cells.

Figure 4. Effect of sodium arsenite on cleaved caspase-8, −9 and −3 and PARP protein expression in MA-10 cells. MA-10 cells were treated without or with sodium arsenite (0.01–100 µM) for 3, 6, 12 and 24 h. Cleaved caspase-8, −9 and −3, and PARP protein expressions were detected by western blotting. (A) Data is representative of one experiment that was further repeated three times. The integrated optical densities of (B) cleaved caspase-8, (C) −9 and (D) −3 and (E) PARP protein expressions were analyzed after normalization to β-actin (43 kDa). Data in B, C, D and E represent the means ± standard error of the mean of three separate experiments. *P<0.05. PARP, poly(ADP-ribose) polymerase.

Figure 5. Effect of dimethylarsenic acid on cleaved caspase-8, −9 and −3 and PARP protein expression in MA-10 cells. MA-10 cells were treated without or with dimethylarsenic acid (0.1 µM-10 mM) for 3, 6, 12, and 24 h. Cleaved caspase-8, −9 and −3, and PARP protein expression was detected by western blotting. (A) Data are representative of one experiment that was further repeated three times. The integrated optical densities of cleaved (B) caspase-8, (C) −9 and (D) −3, and (E) PARP protein expression was analyzed after normalization to β-actin. Data in (B-E) represent the means ± standard error of the mean of three separate experiments. *P<0.05. PARP, poly(ADP-ribose) polymerase.

Caspase inhibitor reverses arsenic-induced MA-10 cell apoptosis

The membrane-permeable and irreversible pancaspase inhibitor Z-VAD-fmk was used to determine whether the cleavage of caspases −3, −8 and −9 could be abolished in arsenicinduced MA10 cell apoptosis. To do so, cells were pretreated with increasing concentrations of Z-VAD-fmk (0.1, 1, 10 and 100 µM) for 2 h, followed by 10 µM sodium arsenite or 1 mM DMA treatment for 24 h. The results from western blotting demonstrated that 10 and 100 µM Z-VAD-fmk treatment significantly reduced caspase-8 cleavage induced by 10 µM sodium arsenite treatment (P<0.05; Fig. 6A and B), compared with 10 µM sodium arsenite treatment alone. In addition, treatment with 0.1–100 µM Z-VAD-fmk significantly reduced caspase-9 protein cleavage (P<0.05; Fig. 6A and C), and treatment with 100 µM Z-VAD-fmk significantly decreased caspase-3 protein activation (P<0.05; Fig. 6A and D), compared with 10 µM sodium arsenite treatment alone. Furthermore, 0.1, 10, and 100 µM Z-VAD-fmk treatment significantly reduced caspase-8 activation (P<0.05; Fig. 7A and B) induced by 1 mM DMA, compared with DMA alone. When used at a concentration range of 0.1–100 µM, Z-VAD-fmk significantly decreased caspase-9 cleavage (P<0.05; Fig. 7A and C) induced by 1 mM DMA, compared with DMA alone. In addition, treatment with 100 µM Z-VAD-fmk significantly decreased caspase-3 cleavage (P<0.05; Fig. 7A and D) induced by 1 mM DMA, compared with DMA alone. These results suggested that the caspase cascades may be crucial in arsenicinduced MA-10 cell apoptosis.

Figure 6. Caspase inhibitor Z-VAD-fmk reverses sodium arsenic-induced caspase cleavage in MA-10 cells. MA-10 cells were pretreated with Z-VAD-fmk for 2 h and co-treated with or without 10 µM sodium arsenite for 24 h. Cleaved caspase-8, −9 and −3 protein expression was detected by western blotting. (A) Data are representative of one experiment that was further repeated three times. The integrated optical densities of cleaved (B) caspase-8, (C) −9 and (D) −3 protein expression were analyzed after normalization to βactin. Data in (B), (C) and (D) represent the mean ± standard error of the mean of three separate experiments. *P<0.05.

Figure 7. Caspase inhibitor Z-VAD-fmk reverses dimethylarsenic acid-induced caspase cascade in MA-10 cells. MA-10 cells were pretreated with Z-VAD-fmk for 2 h and co-treated with or without 1 mM dimethylarsenic acid for 24 h. Cleaved caspase-8, −9 and −3 protein expression was detected by western blotting. (A) Data are representative of one experiment that was further repeated three times. The integrated optical densities of cleaved (B) caspase-8, (C) −9 and (D) −3 protein expression were analyzed after normalization to β-actin. Data in (B), (C) and (D) represent the mean ± standard error of the mean of three separate experiments. *P<0.05.

Discussion

The results from the present study demonstrated that sodium arsenite and DMA induced MA-10 cell apoptosis through caspase pathway activation. The morphological analysis revealed that arsenictreated MA-10 cells became more closely associated with each other and exhibited membrane blebbing, a more rounded appearance and detachment, findings which were consistent with a previous study (15). It has been reported that arsenite blocks the guanosine-5′-triphosphate binding site of tubulin, which leads to microtubule polymerization disruption during mitosis (36). This may explain how arsenic compounds could target the cytoskeleton and cause an enlarged and flattened plasma membrane appearance, which was observed in MA-10 cells from the present study. In addition, the toxicity of arsenic compounds varies, according to the cellular function under consideration. For example, AsO2- can alter the activity of cysteine-rich enzymes by interacting with sulfhydryl groups (37). However, methylated arsenics are metabolites of liver detoxification, which reduces acute arsenic toxicity (38). In the present study, cell viability was significantly decreased to 45% following 12 h treatment with 10 µM sodium arsenite, and to 48% following 12 h treatment with 10 mM DMA. These results demonstrated that sodium arsenite was 1,000-fold more potent than DMA in inducing MA-10 cell toxicity. These findings were comparable with those of other studies, where inorganic arsenite compounds were more toxic than inorganic arsenite compounds (36–38).

Cell cycle regulation is crucial in mammalian cells, and cell cycle checkpoints, including G1 and G2/M, are key regulators that ensure appropriate DNA replication and division (39). The G2/M checkpoint represents the acute response to DNA damage. Subsequently, abnormal G2/M arrest can trigger cell apoptosis (40). It has been reported that arsenite is involved in cell cycle disruption in myelomonocytic leukemia cells (41). In the present study, the cell number in the G2/M phase was significantly increased 24 h following treatment with arsenic compounds. A previous study indicated that arsenic trioxide can induce G2/M arrest through p53 phosphorylation and increased p21 expression in TM4 Sertoli cells (6). Arsenic-induced MA-10 cell apoptosis may therefore be associated with abnormal cell cycle distribution, and it would be interesting to further investigate the mechanism of cell cycle redistribution induced by arsenic compounds in MA-10 cells.

Previous studies have reported that arsenic trioxide induces apoptosis through caspase-9 activation in cultured myeloma (42) and ovarian cancer cells (43). Furthermore, arsenic trioxide induces apoptosis of human keratinocytes via the Fas/FasL pathway (21). In MA-10 cells, caspase-8 and −9 activation was observed following treatment with DMA and sodium arsenite for 12 or 24 h, respectively. In addition, caspase inhibition reversed arsenicinduced MA-10 cell apoptosis. However, caspase-3 cleavage was re-activated when the cleavage of caspase-8 and −9 was inhibited following treatment with 0.1–10 µM Z-VAD-fmk. This observation, at present, is difficult to explain, and therefore merits being investigated in further studies. Treatment with 100 µM Z-VAD-fmk decreased cleavedcaspase-8, −9 and −3 protein levels. These results therefore demonstrated that both arsenic compounds activated the intrinsic and extrinsic apoptotic pathways and induced MA-10 cell apoptosis, which had been reported in previous studies (21,42,43).

Taken together, the results from the present study indicated that arsenic compounds induce apoptosis in vitro. These observations, particularly the higher potency of sodium arsenite, suggest that arsenic compounds may have potential anti-tumorigenic therapeutic application in order to improve the outcome of patients with testicular cancer.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Ministry of Science and Technology (grant no. MOST 105-2320-B-006-001).

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

YFM and YHC contributed to conduct the experiments, figure generations and statistical analysis. MMC designed the experiment and wrote the manuscript. YCC and BMH contributed to experimental design, data interpretation, writing of the manuscript, and ensuring the accuracy and the integrity of the work. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patients consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interest.

References