Abstract
Background/Aim: The effects of inorganic and organic arsenicals on proliferation, cell-cycle distribution, and apoptosis of all-transretinoic acid (ATRA)-resistant human promyelocytic leukemia HL-60 (HL-60-R2) cells were herein investigated. Materials and Methods: Cell proliferation was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell-cycle distribution and apoptotic cells were analyzed by flow cytometry. Results: The 50% inhibitory concentrations (IC50 values) for As2O3 against proliferation of HL-60 and HL-60-R2 cells were 12.2 and 7.2 μM, while those for arsenate were >200 and 62.1 μM, respectively. In contrast, organic methylarsinic acid, dimethylarsonic acid, trimethylarsine oxide, and tetramethylarsonium did not exert any inhibitory effects even at 200 μM. As2O3 and arsenate increased the proportion of apoptotic cells dose-dependently at a concentration range of 5-200 μM. As2O3 did not activate caspase 3/7 in HL-60 and HL-60-R2 cells. Conclusion: As2O3 and arsenate inhibit cell proliferation, affect cell-cycle distribution, and induce apoptosis of ATRA-resistant HL-60-R2 cells. The apoptosis-inducing mechanism appears not to be mediated through caspase3/7.
- Arsenic trioxide
- arsenate
- organic arsenicals
- HL-60 cells
- all-trans-retinoic and resistance
- apoptosis induction
Arsenicals are natural substances that have been used medicinally for over 2,400 years. Recent clinical studies have shown that As2O3 is an effective and relatively safe drug in the treatment of acute promyleocytic leukemia (APL) (1-3). As2O3 is also effective for patients with APL who are resistant to all-transretinoic acid (ATRA) and conventional chemotherapy (4, 5). It has been reported that the effects of As2O3 are not limited to APL cells but can be observed in other cells of either myeloid (6), lymphoid (7) or drug-resistant sublines (8). Anti-proliferative and anti-invasive effects of inorganic and organic arsenic compounds were suggested in human and murine melanoma cells in vitro (9).
The action mechanisms of As2O3 in APL and other malignancies are thought to be due to inhibition of growth and induction of apoptosis (6-8, 10). Glutathione depletion enhances As2O3-induced apoptosis of cancer cells, suggesting that the antineoplastic efficacy of As2O3 relates to an increase in oxidative stress (11-14). Furthermore, selective sensitivity of arsenic compounds to dividing endothelial cells, which results in inhibition of angiogenesis, raise the possibility for designing better targeted-antineoplastic arsenic-containing compounds with less toxicity to normal cells (15). Although mechanisms of the anticancer actions of As2O3 have been studied as described above, the inhibitory effects of As2O3 and many other organic and inorganic arsenic-compounds on ATRA-resistant APL cells have been scarcely studied.
Thus, the present study was undertaken to evaluate anti-proliferative effects of inorganic and organic arsenic compounds on APL-related leukemia cell line HL-60 resistant to ATRA (HL-60-R2) (16), as well as the parental HL-60 cells, and to provide insight into their mechanisms of action by examining cell growth, apoptosis, and caspase 3/7 activity in these cells.
Materials and Methods
Materials. RPMI-1640 medium and fetal bovine serum were purchased from Gibco BRL Co. (Grand Island, NY, USA). As2O3, sodium arsenate, sodium methylarsonate, dimethylarsinic acid, trimethylarsine oxide, and tetramethylarsonium were purchased from Tri Chemical Laboratories Inc. (Yamanashi, Japan). The chemical structures of these arsenic compounds are shown in Figure 1. Stock solutions of these agents were made at a concentration of 10 mmol/l and diluted to working concentrations by distilled water before use. Cell proliferation Kit I and II (MTT) were purchased from Roche Diagnostics Co. (Indianapolis, IN, USA). Annexin V-FITC Apoptosis Detection KitIwere products of BD PharMingen (San Diego, CA, USA).
Cell culture and cell proliferation assay. HL-60-R2 cells were established by exposing parental HL-60 cells to stepwise increasing concentrations (from 100 pM) of ATRA for two months (16). These cell lines were obtained from Riken BioResource Center, Tsukuba, Japan. The leukemia cells were washed and resuspended with the above medium to 5×105 cells/ml, then 98 μl of the cell suspension were placed in each well of a 96-well flat-bottom plate. Two microliters of ethanolic solution containing each test compound or 2 μl of ethanolic solution containing ATRA were added to yield final concentrations of 0.001, 0.01, 0.1, 1, 10 and 100 μmol/l, respectively. Two microliters of ethanol were added to the control wells. The cells were incubated for 72 h in in a humidified chamber with 5%CO2/air at 37°C.
After the incubation period, 15 μl of the MTT labeling reagent (final concentration of 0.5 mg/ml) were added into each well and the plate was mixed on a microshaker for 10 s. The cells were then incubated for a further 4 h in a humidified atmosphere. Subsequently, 100 μl of solubilization solution were added to each well, and the plate was mixed on a microshaker for 10 s and allowed to stand overnight in the incubator in a humidified atmosphere. The spectrophotometrical absorbance of the sample was measured on a microplate reader (Corona MT P-32; Corona Co., Hitachi, Ibaraki, Japan) at 570 nm. A dose–response curve was plotted for each drug, and a concentration which yielded a 50% inhibition of cell growth (IC50, ng/ml) was calculated. The IC50 values were determined from linear regression of at least four points at different concentrations of the agents.
Cell-cycle analysis. HL-60 and HL-60-R2 cells were washed and resuspended with the above medium to 5×105 cells/ml, and then 980 μl of the cell suspension were placed in each well of a 24-well flat-bottom plate. Subsequently, 20 μl of As2O3 solution were added to give final agent concentrations of 0.2-200 μM. After culturing of the cells for 72 h in 5%CO2/air at 37°C, cells were placed in 1.5 ml tubes, washed twice in phosphate-buffered saline containing 1% fetal bovine serum, and then fixed in 70% ethanol at −20°C for 24 h. After fixation, the cells were washed twice with the above buffer, and treated with 1 mg/ml of RNase at 37°C for 30 min. Subsequently, 10 μl of propidium iodide solution (Japan Becton Dickinson, Minato-ku, Tokyo, Japan) was added to this cell suspension (5×105 cells/60 μl), and the tube containing the cell suspension was incubated for 20 min at room temperature in the dark. Subsequently, the cells were resuspended with 800 μl of phosphate-buffered saline containing 0.4% paraformaldehyde and 1% fetal bovine serum, and were analyzed by flow cytometry (Becton Dickinson) within one hour after staining. A total of 30,000 non-gated cells were analyzed.
Caspase-3/7 activity assay. Leukemia cells were incubated with the test agents for 4, 12, and 24 h, respectively. Thereafter, 100 μl of Caspase-Glo 3/7 (Promega, Madison, WI, USA) reagent were added to each sample, and the cells were incubated for 30 min and assayed (8) according to the manufacturer's instructions. The data were analyzed with GraphPad Prism 4.0 (GraphPad Software Inc., La Jolla, CA 92037 USA).
Statistics. Comparison of the data between the two groups was carried-out by unpaired Student's t-test. In each case, p-values less than 0.05 were considered significant.
Results
Effects of inorganic and organic arsenic compounds on cell proliferation of human leukemia cells. Effects of two inorganic and four organic arsenic compounds on cell proliferation and apoptosis of human promyelocytic leukemia HL-60 cells and ATRA-resistant HL-60 (HL-60-R2) cells were examined in vitro. These cells were incubated in the presence of serial concentrations of the agents for 72 h, and then were analyzed with MTT assay procedures.
Decreased sensitivity of HL-60-R2 cells to the growth-inhibitory effects of ATRA, compared to its parental HL-60 cells, was demonstrated, as shown in Figure 2. Statistically significant differences of the effects of ATRA between these two cell lines were observed at concentrations of 0.1, 1, and 10 μM (p<0.05). Inorganic arsenicals, As2O3 and arsenate, dose-dependently inhibited the proliferation of HL-60 cells and HL-60-R2 cells (Figure 3). HL-60-R2 cells were rather sensitive to the growth-inhibitory effects of these inorganic compounds compared to the parental HL-60 cells. Statistically significant differences in the effects of As2O3 and arsenate between these two cell lines were observed at concentrations of 10 μM for As2O3 (p=0.004) (Figure 3A) and 200 μM for arsenate (p<0.001) (Figure 3B), respectively. The IC50 values for As2O3 against the growth of HL-60 cells and HL-60-R2 cells were 12.2, and 7.2 μM, respectively (Table I), while those for arsenate were >200 and 62.1 μM, respectively (Table I). In contrast, the organic compounds methylarsonic acid, dimethylarsinic acid, and trimethylarsine oxide, and tetramethylarsonium did not have any apparent inhibitory effect on HL-60 cells even at 200 μM (Figure 4). We also examined the effects of methylarsonate on the proliferation of HL-60-R2 cells, but this organic compound did not have any suppressive effects, having an IC50 value of more than 20 μM (Table I).
Apoptosis induction and caspase 3/7 activities in leukemia cells treated with inorganic and organic arsenic compounds. As described above, As2O3 and arsenate inhibited proliferation of the parental and ATRAn-resistant HL-60 cells, while the four organic arsenicals did not have any significant effect on the cell proliferation. We further examined the effects of inorganic arsenicals on apoptosis and caspase 3/7 activities in these cell lines. HL-60 cells and HL-60-R2 cells were treated with serial concentrations of As2O3 for 72 h and stained with propidium iodide. Subsequently, cell-cycle analysis was performed with flow cytometry (Figure 5). As2O3 at concentrations of 2-200 μM dose-dependently increased the percentage of apoptotic sub-G1 cells, whereas this agent reduced the percentage of G1 phase cells in both HL-60 and HL-60-R2 cells (Figure 5).
Casepase 3/7 activities in HL-60 and HL-60-R2 cells treated with 10 or 20 μM As2O3 for 4, 12, and 24 h, respectively, are shown in Figure 6. While at these concentrations As2O3 increased the percentage of apoptotic cells as shown in Figure 5, it did not change caspase 3/7 activities in these cells for any incubation time examined (Figure 6).
Discussion
The data described above show that inorganic arsenicals As2O3 and arsenate inhibit proliferation of both HL-60 and the ATRA-resistant HL-60-R2 cells, whereas the four organic arsenicals methylarsonic acid, dimethylarsinic acid, trimethylarsine oxide, and tetramethylarsonium are not effective against the proliferation of HL-60 cells nor HL-60-R2 cells. AIthough the precise mechanism of action of these compounds remains to be elucidated, As2O3 and arsenate are suggested to affect the cell cycle and induce apoptosis in these cell lines without changing caspase 3/7 activities.
We have reported that As2O3 and arsenate have anti-proliferative effects on both T-lymphoblastoid leukemia MOLT-4 cells and daunorubicin-resistant MOLT-4/DNR cells expressing functional P-glycoprotein (8). These observations suggest that As2O3 and arsenate might not be substrates of P-glycoprotein, and thus these agents are effective against multidrug-resistant T-lymphoblastoid leukemia cells expressing functional P-glycoprotein (8). In contrast, the organic arsenicals methylarsonic acid, dimethylarsinic acid, trimethylarsine oxide, and tetramethylarsonium were not effective against the proliferation of MOLT-4 cells and daunorubicin-resistant MOLT-4/DNR cells (8). Suppressive efficacies of As2O3 and arsenate on in vitro proliferation of HL-60 cells were also described by Charoensuk et al. (17), but the effects of these inorganic arsenicals on ATRA-resistant HL-60 cells have not been examined. The HL-60-R2 cell line was established from human promyelocytic leukemia HL-60 cells by exposing the parental HL-60 cells to increasing concentrations of ATRA (16). ATRA is known to inhibit proliferation, promote apoptosis, and induce differentiation of HL-60 cells, whereas HL-60-R2 cells are resistant to all effects of ATRA (16). The molecular mechanism underlying the ATRA resistance in HL-60-R2 cells, however, is not well understood. It is, therefore, noteworthy that the anti-proliferative effects of As2O3 at 20 μM and arsenate at 200 μM are significantly greater on the ATRA-resistant HL-60-R2 cells than on the parental HL-60 cells in the present study (Figure 3). When we compare the IC50 values of these inorganic arsenicals, the effects on the resistant HL-60-R2 cells were 1.69- to 3.22-times greater than those on the parental HL-60 cells (Table I). These results suggest that As2O3 is useful for the patients with APL showing resistance to ATRA therapy, which is actually relevant to the clinical efficacy of As2O3. Studies on clinical pharmacokinetics of As2O3 have shown that the peak level of plasma As2O3 concentration under a general treatment schedule was 5.5 to 7.3 μM (18). The IC50 values for As2O3 against the proliferation of HL-60 cells and HL-60-R2 cells were 12.21 and 7.22 μM, respectively (Table I), which are near peak plasma levels after administration of the agent to patients with leukemia.
Our present study suggests that As2O3 affects the cell cycle and induces apoptosis of both HL-60 and HL-60-R2 cells, probably via a cellular signaling pathway independent of caspase 3/7. We have reported that As2O3 influences the cell cycle and induces apoptosis of MOLT-4 and MOLT-4/DNR cells via activation of caspase-3 (11). These discrepant effects of As2O3 on caspase-3 induction might be due to the difference in leukemia cell lines. Charoensuk et al. reported that an increase in caspase-3 activity in HL-60 cells was observed by treatment of cells with certain organic arsenicals (17), other than those we examined in this study. These data imply that certain kinds of organic arsenicals induce apoptosis of HL-60 cells through a caspase-dependent mechanism. They also showed that As2O3 and arsenate failed to activate caspase-3, suggesting that these inorganic arsenicals induce apoptosis by an alternative pathway (17). Our present data are consistent with their observations, and thus further studies are required for disclosing the molecular mechanism of the apoptosis-inducing effect of these inorganic arsenicals in ATRA-resistant HL-60-R2 cells. As2O3 has been reported to induce oxidative stress, DNA damage, and the mitochondrial pathway of apoptosis in several human cancer cell lines (19-21). These observations suggest that As2O3 induces apoptosis of ATRA-resistant HL-60-R2 cells through similar mechanisms related to oxidative stress induction.
The present study, in summary, showed that inorganic arsenic compounds, As2O3 and arsenate, inhibit cell proliferation, affect the cell cycle, and induce apoptosis of promyelocytic leukemia HL-60 cells and the ATRA-resistant HL-60-R2 cells. In contrast, organic compounds methylarsonic acid, dimethylarsinic acid, trimethylarsine oxide, and tetramethylarsonium did not have any inhibitory effects on these cells. The apoptosis-inducing mechanism of As2O3 appears not to be mediated through caspase3/7, although the precise action mechanism of As2O3 remains to be elucidated.
Footnotes
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Conflicts of Interest
The Authors declare that there are no conflicts of interest in this study.
- Received July 9, 2014.
- Revision received August 13, 2014.
- Accepted August 19, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved