Contributed equally
Arsenic is a well-documented environmental toxicant that can induce neurotoxicity and peripheral vascular diseases. In fact, arsenic trioxide has been used to treat various cancer types. Oral cancer has been in the top ten common cancers for decades in Taiwan, and the incidence rate is continuously increasing. The majority of oral cancers are associated with excessive tobacco, alcohol consumption and betel chewing. To the best of our knowledge, no study has revealed the effect of arsenic compounds on oral cancers. Thus, the present study used OEC-M1 oral squamous carcinoma cells treated with sodium arsenite (NaAsO2) and dimethylarsenic acid (DMA) to determine whether both arsenic compounds could exert anticancer effects on oral cancer. The results demonstrated that NaAsO2 and DMA induced rounding up and membrane blebbing in OEC-M1 cells, which are morphological characteristics of apoptosis. Annexin V/PI double staining analysis further confirmed that both arsenic compounds induced apoptosis of OEC-M1 cells. In addition, NaAsO2 and DMA significantly decreased the survival rate and increased the percentage of OEC-M1 cells in the subG1 and G2/M phases (P<0.05). Furthermore, both arsenic compounds significantly activated the cleavage of caspase-8, −9, −3 and PARP, and the phosphorylation of JNK, ERK1/2 and p38 in OEC-M1 cells (P<0.05). Collectively, the findings of the present study indicated that NaAsO2 and DMA stimulate extrinsic and intrinsic apoptotic pathways through the activation of the MAPK pathways to induce apoptosis of OEC-M1 cells, suggesting that NaAsO2 and DMA may be used as novel anticancer drugs for oral cancers.
The most frequent malignant tumor of the head and neck region is squamous cell carcinoma, which typically develops in males (
Arsenic is a naturally occurring element distributed throughout the earth's crust, which exhibits both metallic and non-metallic properties (
The unifying characteristics of apoptosis are largely morphological, including cell shrinkage, blebbing of the plasma membrane, chromatin condensation and DNA fragmentation (
Mitogen-activated protein kinases (MAPKs), which are crucial for the maintenance of cell proliferation, differentiation, mitosis, cell survival, gene expression and apoptosis, consist of three family members: c-Jun NH2-terminal kinase [(JNK)1, 2 and 3]; extracellular signal-regulated kinase (ERK1 and 2); and p38 MAPKs (p38-MAPK α, β, γ and δ) (
On account of the anticancer ability of arsenic compounds and emerging demands for more effective therapeutic remedies to treat oral cavity cancer, OEC-M1 cells, derived from a surgical specimen of buccal mucosa squamous carcinoma from a Taiwanese as a unique indigenous oral cancer cell line (
NaAsO2 was purchased from Fluka; Sigma-Aldrich. DMA, high glucose Dulbecco's modified Eagle's medium, penicillin-streptomycin, staurosporine, RNase A, propidium iodide, 30% acrylamide/Bis-acrylmide solution, methylthiazole tetrazolium (MTT) and monoclonal antibody against β-actin were purchased from Sigma-Aldrich; Merck KGaA. Fetal bovine serum and trypsin-EDTA were purchased from AG Scientific, Inc. RPMI-1640 medium, Dulbecco's modified Eagle's medium/F12 and Keratinocyte-SFM medium were purchased from Gibco; Thermo Fisher Scientific, Inc. Sodium chloride, potassium chloride, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and Tris base were purchased from JT Baker. Disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4) and sodium bicarbonate (NaHCO3) were purchased from Riedel-de Haen. Hydrochloric acid, sodium hydroxide, sodium dodecyl sulfate (SDS), Tween-20 and dimethyl sulfoxide (DMSO) were purchased from Merck KGaA. Donkey anti-rabbit IgG conjugated to horseradish peroxidase and donkey anti-mouse IgG conjugated to horseradish peroxidase were purchased from PerkinElmer, Inc. Annexin V-FITC apoptosis detection kit was purchased from Strong Biotech. Micro BCA protein assay kit was purchased from Thermo Fisher Scientific, Inc. Antibodies against PARP, cleaved caspase-8, cleaved caspase-9, cleaved caspase-3, phosphorylated (p)-JNK, JNK, p-ERK1/2, ERK1/2, p-p38 and p38 were purchased from Cell Signaling Technology, Inc. Enhanced chemiluminescence (ECL) detection kit was purchased from EMD Millipore.
OEC-M1 is a cell line derived from a surgical specimen of buccal mucosa squamous carcinoma from a Taiwanese, a unique oral cancer indigenous in Taiwan, and was a generous gift from Professor Kuo-Wei Chang (National Yang-Ming University, Taipei, Taiwan) (
OEC-M1 cells were seeded at a concentration of 6×105 cells in a 6-cm Petri dish with 2 ml culture medium. After reaching 70–80% confluence, cells were treated without or with various concentrations of NaAsO2 (0.1, 1, 10, 25, 50 and 100 µM), or various concentrations of DMA (0.1, 1, 2, 5, 10, 25, 50 and 100 mM) for 24 h. Cell morphology was then observed under an Olympus CK40 light microscope and recorded by an Olympus DP20 digital camera (Olympus Corporation).
OEC-M1 cells were seeded at a concentration of 1×104 cells/well with 100 µl culture medium. After reaching 70–80% confluence, cells were treated without or with various concentrations of NaAsO2 (0.1, 1, 10, 25, 50 and 100 µM) or various concentrations of DMA (0.1, 1, 10, 25, 50 and 100 mM) for 24 h. MTT was added to a final concentration of 0.5 mg/ml and incubated at 37°C for 4 h. The medium was discarded and 50 µl DMSO was added to each well to dissolve the crystals by shaking the plate for 20 min in the dark (
To further confirm whether NaAsO2 and DMA could induce apoptosis among these oral cancer cell lines, the redistribution of the cell cycle was examined by flow cytometric analysis through propidium iodide (PI) staining. OEC-M1 cells were seeded at a concentration of 6×105 cells. After reaching 70–80% confluence, cells were treated without or with various concentrations of NaAsO2 (0.1, 1, 10, 25, 50 and 100 µM), or various concentrations of DMA (0.1, 1, 10, 25, 50 and 100 mM) for 24 h. Treated cells were harvested by trypsin and centrifuged (400 × g for 12 min at 4°C), and then washed by isoton II and fixed in 70% ethanol at −20°C for ≥2 h. After fixation, cells were washed with isoton II again and collected by centrifugation (400 × g for 12 min at 4°C). Cell pellets were resuspended with isoton II and mixed with 100 µg/ml RNase and 40 µg/ml PI for 30 min (
Treated cells were harvested by trypsin and washed with 2 ml culture medium. Following centrifugation at 160 × g for 10 min at 4°C, the pellets were resuspended with cold isoton II and centrifuged (400 × g for 12 min at 4°C) again. The pellets were subsequently mixed with 100 µl staining solution for 15 min according to the user's manual of the Annexin V-FITC apoptosis detection kit (Strong Biotech). The stained cells were analyzed at a wavelength of 488 nm excitation using a 515-nm band pass filter for FITC detection and >600-nm band pass filter for PI detection by a FACScan flow cytometer (BD Biosciences). The plots were divided into four quadrants, which represent negative staining (viable) cells, Annexin V-positive (early apoptosis) cells, PI-positive (necrosis) cells, and Annexin V/PI double-positive (late apoptosis) cells. In addition, cells were also treated with staurosporine which was considered as a positive control. The percentage of cells in the 4 quadrants was analyzed using FACStation v6.1× software.
Cells (6×105) were seeded in a 60-mm dish. After reaching 70–80% confluence, cells were treated without or with various concentrations of NaAsO2 (10 and 25 µM), or various concentrations of DMA (10 and 25 mM) for 3, 6, 12 and 24 h. Medium was then transferred to a 15-ml tube, and centrifuged at 1,500 × g for 10 min at 4°C. Attached cells were lysed using 100 µl lysis buffer with proteinase inhibitor (cat. no. P8340; Sigma-Aldrich; Merck KGaA). The pellets were resuspended with 10 µl lysis buffer, blended into cell lysates, and centrifuged at 12,000 × g for 12 min at 4°C. The supernatants were collected and stored at −80°C. The protein concentrations of cell lysates were determined by the Lowry assay (
For immunoblotting analysis, cell lysates (30 µg) were resolved on 12% SDS-PAGE gel with standard running buffer at room temperature, and electrophoretically transferred to polyvinyldifluoride membranes at 4°C. The membranes were blocked in 4% milk for 1 h at room temperature and incubated with primary antibodies [cleaved caspase-8 (product no. 9429; 1:1,000), cleaved caspase-9 (product no. 9509; 1:1,000), cleaved caspase-3 (product no. 9661; 1:1,000), cleaved PARP (product no. 9542; 1:1,000), phospho-JNK (product no. 9251; 1/4,000), JNK (product no. 9252; 1:1,000), phospho-ERK1/2 (product no. 9101; 1:4,000), ERK1/2 (product no. 9102; 1:4,000), phospho-p38 (product no. 9215; 1:1,000), p38 (product no. 9212; 1:4,000)] overnight at 4°C. Following washing with TBS Tween-20 and incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature [Donkey anti-rabbit IgG; cat. no. NEF81200-1EA; 1:2,000], the membranes were visualized using the ECL detection kit and UVP EC3 BioImaging Systems (UVP; Analytik Jena) (
The data are expressed as the mean ± standard error of the mean (SEM) of at least three independent experiments. The statistical significance of differences between the control and treatment groups was determined by one-way ANOVA, followed by Tukey's post hoc test comparisons. Statistical analysis was performed with GraphPad Prism 6 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
OEC-M1 cells were treated with medium, NaAsO2 (trivalent arsenic) (0.1, 1, 10, 25, 50 and 100 µM) or DMA (penta-valent arsenic) (0.1, 1, 10, 25, 50 and 100 mM) for 24 h, and the morphological changes were examined. In the experiment with NaAsO2, OEC-M1 cells exhibited a spindle-like shape in the control group (
The survival rate of OEC-M1 cells treated with arsenic compounds was further examined by MTT cell viability assay. Cells were treated with medium, NaAsO2 (0.1, 1, 10, 25, 50 and 100 µM) or DMA (0.1, 1, 10, 25, 50 and 100 mM) for 24 h. The OEC-M1 cell survival rate was significantly decreased by NaAsO2 at doses of 10–100 µM (
To assess whether NaAsO2 and DMA could induce OEC-M1 oral cancer cell death via apoptosis, cells were treated with arsenic compounds and then evaluated for their DNA contents by PI staining via flow cytometric analysis. Thus, OEC-M1 cells were treated with medium, NaAsO2 (0.1, 1, 10, 25, 50 and 100 µM) or DMA (0.1, 1, 10, 25, 50 and 100 mM) for 24 h, and the changes in the number of cells in the subG1, G1 and G2/M phases related to cell cycle regulation were investigated.
NaAsO2 at 50 and 100 µM significantly increased the number of cells in the subG1 phase in OEC-M1 cells (
To further confirm whether apoptosis is induced by arsenic compounds in OEC-M1 cells, double staining with Annexin V and PI via flow cytometer was performed. The percentages of double-negative (viable), PI-positive (necrotic), Annexin V-positive (early apoptotic) and double-positive (late apoptotic) cells can be revealed in four quadrants by the double staining assay to assess cell apoptosis (
To further investigate whether arsenic compounds can induce OEC-M1 cell apoptosis via the death receptor (extrinsic) and/or mitochondrial (intrinsic) apoptotic pathways, the expression levels of cleaved caspase-8 and caspase-9, and the downstream targets of cleaved caspase-3 and PARP were examined by western blotting (
NaAsO2 at 10 and 25 µM for 12 and 24 h significantly activated caspase-8 (
Previous studies have revealed that MAPK pathways may be involved in regulating cell proliferation, differentiation, mitosis, cell survival, gene expression and apoptosis (
NaAsO2 at 10 µM for 24 h and 25 µM for 3, 6 and 24 h could significantly increase the level of p-JNK (
In the present study, NaAsO2 (inorganic arsenic) and DMA (organic arsenic) both demonstrated the ability to induce apoptosis of OEC-M1 oral cavity cancer cells. Studies have revealed that the cytoskeleton may be a target of NaAsO2 and DMA (
Inorganic arsenic is significantly more toxic compared with organic arsenic compounds (
The eukaryotic cell cycle proceeds to the next phase as the upstream events fulfill the requirements of checkpoints (
Apoptosis is a critical process for maintaining homeostasis (
In fact, studies have reported that arsenic compounds can activate different signaling pathways to induce cell death related to apoptotic pathways among different cancer cell types, such as myeloma cells, neutrophils and/or leukemia (
Apoptotic pathways are associated with numerous regulating mechanisms, one of which is the MAPK signaling pathway responding to cellular stress. MAPK signaling may either promote survival or enhance sensitivity to apoptosis depending on the cell types, stimuli and the latency of the activation of MAPKs (
In the present study, both NaAsO2 and DMA could induce cell apoptosis through extrinsic and intrinsic apoptotic pathways, exerting potential antitumor effects on OEC-M1 oral cancer cells. Furthermore, both arsenic compounds were able to induce phosphorylation of JNK, ERK and p38 MAPKs to modulate the activation of the caspase cascade to stimulate apoptosis of OEC-M1 cells. However, the precise roles upon the activation of MAPK and caspase pathways by NaAsO2 and DMA in OEC-M1 cells remain elusive, since specific inhibitors and/or siRNA were not used in the present study to block apoptosis. With the use of specific inhibitors and/or siRNA to suppress OEC-M1 cell apoptosis, the activation of MAPK and caspase pathways by NaAsO2 and DMA in OEC-M1 cells can then be confirmed, which will conclusively reveal the underlying molecular mechanism. In addition, studies have demonstrated that intravenous arsenic trioxide and its derivatives both alone and/or in combination with other agents have been used successfully for the treatment of APL and myeloid neoplasms, and arsenic trioxide is a standard treatment for APL patients to date (
It should be noted that the error bars are rather sizable in certain figures, regarding the western blot analysis of caspase and MAPK protein expression activated by NaAsO2 and DMA in OEC-M1 cells. It is well known that in
Not applicable.
The present study was supported by the
The data that support the findings of this study are available from the corresponding author upon reasonable request.
NPF, CLK and CYC designed the experiments, carried out the experiments, interpreted the results, and wrote the manuscript. CYW, ECS and BMH participated in the design and coordination of the study, were involved in the statistical analysis of the obtained results and were responsible for the revision of the manuscript for substantive and methodological correctness. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Effects of arsenic compounds on morphological changes in OEC-M1 cells. Cells were treated with (A) plain medium, (B-G) 0.1, 1, 10, 25, 50 and 100 µM sodium arsenite, respectively, or (H-M) 0.1, 1, 10, 25, 50 and 100 mM dimethylarsenic acid in OEC-M1 cells for 24 h, respectively. Morphological changes were observed under light microscopy (scale bar, 50 µm). Arrowheads indicate membrane-blebbing cells. (N) Apoptotic cell (scale bar, 10 µm). The arrowhead indicates an apoptotic cell with membrane blebbing). Experiments were performed three times with similar results.
Effects of arsenic compounds on cell viability in OEC-M1 cells. Cells were treated with (A) 0, 0.1, 1, 10, 25, 50 and 100 µM NaAsO2, or (B) 0, 0.1, 1, 10, 25, 50 and 100 mM DMA for 24 h. Cell viability was quantified by MTT assay. Results are expressed as percentages of cell growth relative to the control groups as 100%. The data are expressed as mean ± SEM of at least three separate experiments. **P<0.01 and ***P<0.001 represent statistical differences compared to the Ctrl. NaAsO2, sodium arsenite; DMA, dimethylarsenic acid; SEM, standard error of the mean; Ctrl, control.
Effects of NaAsO2 on cell cycle redistribution in OEC-M1 cells. (A) Various concentrations of NaAsO2 (0, 0.1, 1, 10, 25, 50 and 100 µM) were used to treat OEC-M1 for 24 h. Cells were fixed, stained with PI, and analyzed by flow cytometry. Cells in subG1 phase contain less DNA content than normal cells, indicating apoptosis. The percentage of (B) sub G1, (C) G1 and (D) G2/M phase cells are presented, respectively. The data are expressed as the mean ± SEM of at least three separate experiments. *P<0.05, **P<0.01, and ***P<0.001 represent statistical differences compared to the Ctrl group. NaAsO2, sodium arsenite; PI, propidium iodide; SEM, standard error of the mean; Ctrl, control.
Effects of DMA on cell cycle redistribution in OEC-M1 cells. (A) Various concentrations of DMA (0, 0.1, 1, 10, 25, 50 and 100 mM) were used to treat OEC-M1 for 24 h. Cells were fixed, stained with PI, and analyzed by flow cytometry. Cells in subG1 phase contain less DNA content than normal cells, indicating apoptosis. The percentage of (B) sub G1, (C) G1 and (D) G2/M phase cells are presented, respectively. The data are expressed as the mean ± SEM of at least three separate experiments. *P<0.05 and ***P<0.001 represent statistical differences compared to the Ctrl group. DMA, dimethylarsenic acid; SEM, standard error of the mean; PI, propidium iodide; Ctrl, control.
NaAsO2 induces OEC-M1 cell apoptosis. Various concentrations of NaAsO2 (0, 0.1, 1, 10, 25, 50 and 100 µM) were used to treat OEC-M1 for 24 h. (A) The apoptotic status was determined by Annexin V/PI double staining assay, and the staurosporine-treated cells were considered as a positive control. (B) The percentages of double-negative (viable), PI single-positive (necrotic), Annexin V single-positive (early apoptotic), and double-positive (late apoptotic) cells are presented in. (C) Annexin V-positive cells were analyzed and are presented. The data are expressed as the mean ± SEM of at least three separate experiments. *P<0.05 and ***P<0.001 represent statistical differences compared to the Ctrl group. NaAsO2 sodium arsenite; SEM, standard error of the mean; PI, propidium iodide; Ctrl, control.
DMA induces OEC-M1 cell apoptosis. Various concentrations of DMA (0, 0.1, 1, 10, 25, 50 and 100 mM) were used to treat OEC-M1 for 24 h. (A) The apoptotic status was determined by Annexin V/PI double staining assay, and the staurosporine-treated cells were considered as a positive control. (B) The percentages of double-negative (viable), PI single-positive (necrotic), Annexin V single-positive (early apoptotic), and double-positive (late apoptotic) cells are presented. (C) Annexin V-positive cells were analyzed and are presented. The data are expressed as the mean ± SEM of at least three separate experiments. ***P<0.001 represent statistical differences compared to the Ctrl group. DMA, dimethylarsenic acid; SEM, standard error of the mean; PI, propidium iodide; Ctrl, control.
Effects of NaAsO2 on the expression levels of cleaved caspase-8, −9, −3 and PARP proteins in OEC-M1 cells. Cells were treated without or with 10 and 25 µM NaAsO2 for 3, 6, 12 and 24 h, respectively. (A) Cleaved caspase-8 (43 kDa), −9 (35/37 kDa), −3 (17/19 kDa) and PARP (~85-90 kDa) were detected by western blotting. The IODs of cleaved (B) caspase-8, (C) caspase-9, (D) caspase-3 and (E) PARP proteins were normalized with β-actin (43 kDa) in each lane. The data are expressed as the mean ± SEM of at least three separate experiments. *P<0.05, **P<0.01, and ***P<0.001 represent statistical differences compared to the Ctrl group. NaAsO2, sodium arsenite; PARP, poly(ADP-ribose) polymerase; IODs, integrated optical densities; SEM, standard error of the mean; C or Ctrl, control.
Effects of DMA on the expression levels of cleaved caspase-8, −9, −3 and PARP proteins in OEC-M1 cells. Cells were treated without or with 1 and 10 mM DMA for 3, 6, 12 and 24 h, respectively. (A) Cleaved caspase-8 (43 kDa), −9 (35/37 kDa), −3 (17/19 kDa) and PARP (~85-90 kDa) were detected by western blotting. The IODs of cleaved (B) caspase-8, (C) caspase-9, (D) caspase-3 and (E) PARP proteins were normalized with β-actin (43 kDa) in each lane. The data are expressed as the mean ± SEM of at least three separate experiments. *P<0.05, **P<0.01, and ***P<0.001 represent statistical differences compared to the Ctrl group. DMA, dimethylarsenic acid; PARP, poly(ADP-ribose) polymerase; IODs, integrated optical densities; SEM, standard error of the mean; C or Ctrl, control.
Effects of NaAsO2 on the phosphorylation of the MAPK signaling pathway in OEC-M1 cells. Cells were treated without or with 10 and 25 µM NaAsO2 for 3, 6, 12 and 24 h, respectively. (A) p-JNK (46/54 kDa), JNK, p-ERK1/2 (42/44 kDa), ERK1/2, p-p38 (43 kDa) and p38 were detected by western blotting. The IODs of (B) p-JNK, (C) p-ERK and (D) p-p38 proteins were normalized with total forms of themselves in each lane. The data are expressed as the mean ± SEM of at least three separate experiments. *P<0.05, **P<0.01, and ***P<0.001 represent statistical differences compared to the Ctrl group. NaAsO2, sodium arsenite; MAPK, mitogen-activated protein kinase; p-, phosphorylated; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; IODs, integrated optical densities; SEM, standard error of the mean; C or Ctrl, control.
Effects of DMA on the phosphorylation of the MAPK signaling pathway in OEC-M1 cells. Cells were treated without or with 1 and 10 mM DMA for 3, 6, 12 and 24 h, respectively. (A) p-JNK (46/54 kDa), JNK, p-ERK1/2 (42/44 kDa), ERK1/2, p-p38 (43 kDa) and p38 were detected by western blotting. The IODs of (B) p-JNK, (C) p-ERK and (D) p-p38 proteins were normalized with total forms of themselves in each lane. The data are expressed as the mean ± SEM of at least three separate experiments. **P<0.01 and ***P<0.001 represent statistical differences compared to the Ctrl group. DMA, dimethylarsenic acid; MAPK, mitogen-activated protein kinase; p-, phosphorylated; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; IODs, integrated optical densities; SEM, standard error of the mean; C or Ctrl, control.