Introduction
Arsenic trioxide (As2O3) has shown substantial efficacy in the treatment of patients with acute promyelocytic leukemia (APL), a specific subtype of acute myeloid leukemia (AML) (1). Previous studies have demonstrated that As2O3 influences various intracellular signaling pathways, which may result in the induction of apoptosis, the inhibition of growth and angiogenesis, and the promotion of differentiation (2). In clinical studies, the complete remission (CR) rate of As2O3 treatment was substantial (3–5). However, at the same time, a significant portion of patients could not achieve CR in spite of the treatment using As2O3 as well as all-trans-retinoic acid (ATRA) and conventional chemotherapy. Hence, it is necessary to investigate a novel treatment method overcoming the resistance of current treatments.
The 1,25-dihydroxyvitamin D3 (also known as 1,25(OH)2D3 or calcitriol), the active form of vitamin D, is also known to regulate cell proliferation and differentiation as well as classical actions on calcium homeostasis (6–8). 1,25(OH)2D3 was found to cause differentiation of myeloid leukemic cells and to prolong the survival of leukemic mice (9,10). In addition, previous studies have demonstrated that 1,25(OH)2D3 causes differentiation of the chemo-naïve APL cell line HL-60 (11,12) and the ATRA-resistant APL cell line UF-1 (13). However, in a previous clinical study performed in patients with myelodysplastic syndrome, 1,25(OH)2D3 single therapy resulted in hypercalcemia in half of the patients before concentrations necessary for sufficient anti-leukemic activity could be achieved (14). This hypercalcemic side effect has limited the clinical application of 1,25(OH)2D3 single therapy to hematologic malignancies.
In this study, we aimed to elucidate the anti-leukemic effect of 1,25(OH)2D3 combined with As2O3 on human AML cells. However, considering the dose-dependent hypercalcemic effect of 1,25(OH)2D3, we used a low-dose of 1,25(OH)2D3. Thus, the objective of this study was to elucidate the anti-leukemic effect of low-dose 1,25(OH)2D3 combined with As2O3 on HL-60 and K562 cells.
Materials and methods
Cell culture
The HL-60 and K562 cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea). HL-60 and K562 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 units of penicillin and 100 μg/ml of streptomycin in a 37°C incubator under 5% CO2.
Measurement of cytotoxicity with trypan blue exclusion test
Inhibition of the proliferation rate of HL-60 and K562 cells was measured by the trypan blue exclusion test. The cytotoxic effect of As2O3 on HL-60 and K562 cells was examined by treating 2.4×105 cells with 0, 0.5, 1.0, 1.5, 2.0 and 3.0 μM As2O3 for 24, 48 and 72 h. The cytotoxic effect of 1,25(OH)2D3 on HL-60 and K562 cells was examined by treating 2.5×104 cells with 0, 100, 200, 300, 400 and 500 μM As2O3 for 24, 48 and 72 h. The effect of the combination treatment on HL-60 cells was evaluated by treating 1.2×106 cells for 24 h with the following combinations: 0.5 μM As2O3 plus 50 nM 1,25(OH)2D3, 1.0 μM As2O3 plus 100 nM 1,25(OH)2D3, 1.5 μM As2O3 plus 150 nM 1,25(OH)2D3, 2.0 μM As2O3 plus 200 nM 1,25(OH)2D3 and 3.0 μM As2O3 plus 300 nM 1,25(OH)2D3. The effect of the combination treatment on K562 cells was evaluated by treating 1.2×106 cells for 24 h with the following combinations: 0.25 μM As2O3 plus 25 nM 1,25(OH)2D3, 0.50 μM As2O3 plus 50 nM 1,25(OH)2D3, 0.75 μM As2O3 plus 75 nM 1,25(OH)2D3, 1.00 μM As2O3 plus 100 nM 1,25(OH)2D3 and 1.50 μM As2O3 plus 150 nM 1,25(OH)2D3.
After treatment, we diluted the cells in complete medium, without FBS, to an approximate concentration of 1–2×105 cells/ml. Then, we added 0.1 ml of 0.4% trypan blue solution to 0.5 ml of the cell suspensions and mixed them thoroughly. These mixtures were incubated for 5 min at 15–30°C. The cell count was calculated using a hemocytometer (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) and a microscope (CKX41; Olympus, Tokyo, Japan). Each experiment was performed in duplicate.
mRNA extraction and RT-PCR
HL-60 and K562 cells were treated for 24 h with the following combinations: control; 100 nM 1,25(OH)2D3; 1.0 μM As2O3; 100 nM 1,25(OH)2D3 plus 1.0 μM As2O3; and 100 nM 1,25(OH)2D3 plus 3.0 μM As2O3. Total RNA was isolated from the cells using TRIzol reagent. The RNA pellets obtained were dissolved in diethylpyrocarbonate (DEPC)-treated H2O at concentrations of 0.5 to 1.0 μg/μl, and then stored at −70°C. The quantity and quality of the RNA preparations were determined by measuring the absorbance at 260 and 280 nm. One microgram of total RNA was reverse-transcribed using a first strand cDNA synthesis kit with random primer p(dN)6 and the primers listed in Table I. The amplification conditions used were 35 cycles at −94°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. All data were normalized to the internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Western blot analysis
As2O3- or 1,25(OH)2D3-treated HL-60 and K562 cells were prepared in the same way as described for the RT-PCR analysis. These cells were homogenized in 10 mM Tris (pH 7.4), 1 mM sodium vanadate (Na3VO4) and 1% sodium dodecyl sulfate (SDS). These homogenates were boiled for 5 min at 95°C and centrifuged at 13,000 × g for 15 min at 4°C. The pellet was discarded and the supernatant containing the protein was transferred to a clean tube. The total protein concentration was measured using the micro-bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA) in accordance with manufacturer's instructions. Samples containing 30 μg of protein, along with the molecular weight marker (BenchMark™ Pre-Stained Protein Ladder; Invitrogen, Grand Island, NY, USA), were subjected to 10% SDS polyacrylamide gel electrophoresis under reducing conditions. The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After nonspecific sites were blocked with 5% powdered skim milk in 0.05% Triton X-100/Tris-buffered saline (TBS-T) for 1 h, blots were incubated overnight with an IgG-purified rabbit polyclonal Bcl-2, Bax, or caspase-3 antibody (Cell Signaling Technology, Danvers, MA, USA) in a solution containing 5% powdered skim milk and 0.05% Triton X-100/TBS. The blots were then washed three times in TBS-T for 10 min each and incubated with a peroxidase-conjugated goat anti-rabbit IgG at a concentration of 1 μg/ml in 5% powdered skim milk in 0.05% TBS-T. All samples were also blotted for β-actin (clone AC-15; Sigma-Aldrich, Dublin, Ireland) to normalize protein amounts.
Evaluation of apoptosis using flow cytometry
As2O3- or 1,25(OH)2D3-treated HL-60 cells were prepared similarly as described in the RT-PCR analysis. The cells were washed twice with cold PBS and then resuspended in 1X binding buffer at a concentration of 2×105 cells/ml. A total of 100 μl of the mixtures was then transferred to a 5-ml culture tube and then 5 μl of fluorescein-conjugated Annexin V (Annexin V-FITC) and 5 μl of propidium iodide (PI) were added to the culture tube. The cells were gently vortexed and incubated for 15 min at room temperature in the dark, and 150 μl of binding buffer was added to each tube. Flow cytometry was then performed within 1 h.
Statistical analysis
The combined effects of As2O3 and 1,25(OH)2D3 were analyzed by CalcuSyn software (Biosoft, Ferguson, MO, USA) using the Chou-Talalay method (15). This method is based on the median-effect equation for a dose-effect relationship fa/fu = (D/Dm)m, where D is the dose, Dm is the IC50, fa is the fraction affected by dose D, fu is the unaffected fraction (fu = 1 - fa) and m is a coefficient of the sigmoidicity of the dose-effect curve. The combination index (CI) was determined on the basis of the isobologram analysis for mutually exclusive effects: CI = (D)1/(Dx)1 + (D)2/(Dx)2, where (Dx)1 and (Dx)2 are the concentrations of As2O3 and 1,25(OH)2D3 which inhibit cell growth by x% and (D)1 and (D)2 are the drug concentrations in the combination treatments which inhibit cell growth by x%. (Dx)1 and (Dx)2 values can be determined by a rearrangement of equation D = Dm (fa/(1- fa))1/m. The CI values of <1, 1 and >1 indicate synergism, an additive effect, and antagonism, respectively.
Results
Synergistic cytotoxic effect of the combination treatment of As<sub>2</sub>O<sub>3</sub> and low-dose 1,25(OH)<sub>2</sub>D<sub>3</sub>
As shown in Fig. 1, the viability of HL-60 and K562 cells was in reverse proportion to the concentration of As2O3 (0, 0.5, 1.0, 1.5, 2.0 and 3.0 μM) or 1,25(OH)2D3 (0, 100, 200, 300, 400 and 500 nM). K562 cells were more sensitive to both As2O3 and 1,25(OH)2D3 than HL-60 cells.
The results of the combination treatment with both As2O3 and low-dose 1,25(OH)2D3 are summarized in Table II and III. In both HL-60 and K562 cells, the combination treatment with As2O3 and 1,25(OH)2D3 at a 10:1 ratio revealed a more prominent cytotoxicity compared to single treatments with either As2O3 or 1,25(OH)2D3. Additionally, as shown in Fig. 2, in both HL-60 and K562 cells, the CI values of all combinations evaluated were <1, which suggests evident synergistic cytotoxic effects for As2O3 and low-dose 1,25(OH)2D3.
Expression of apoptosis-related genes (mRNA)
The expression of apoptosis-related genes in HL-60 and K562 cells was analyzed using the RT-PCR method (Fig. 3). In both HL-60 and K562 cell lines, decreased Bcl-2 and increased Bax and caspase-3 expression was observed in either As2O3- or 1,25(OH)2D3-treated cells compared to the control. In addition, combination treatment using both As2O3 and 1,25(OH)2D3 more prominently decreased Bcl-2 expression and increased Bax and caspase-3 expression. Additionally, the effect of the combination treatment was enhanced in proportion to the increased concentration of As2O3; 3 μM As2O3-treated cells showed decreased Bcl-2 and increased Bax and caspase-3 expression when compared to the expression in 1 μM As2O3-treated cells.
Expression of apoptosis-related proteins
Western blot analysis was performed to analyze the expression of apoptosis-related proteins in the HL-60 and K562 cells. As shown in Fig. 4, the results for both HL-60 and K562 cell lines were identical to those of the RT-PCR analysis (Fig. 3). The combination treatment enhanced the production of pro-apototic Bax and caspase-3 proteins and reduced the production of anti-apoptotic Bcl-2 protein. In addition, the effect of the combination treatment was enhanced in proportion to the increased concentration of As2O3.
Enhancement of As<sub>2</sub>O<sub>3</sub> and 1,25(OH)<sub>2</sub>D<sub>3</sub>-induced apoptosis
HL-60 cells were labeled with Annexin V-FITC and PI, and analyzed using flow cytometry to differentiate whether the main cause of cell death was apoptosis or necrosis. As shown in Fig. 5, cell death was significantly increased after the combination treatment with As2O3 and 1,25(OH)2D3 in the HL-60 cells. The proportion of living cells (Annexin V-FITC- and PI-negative) was 92.6% in the control; 51.3% in the cells treated with 1.0 μM As2O3; 86.4% in the cells treated with 100 nM 1,25(OH)2D3; and 2.0% in the cells treated with 1.0 μM As2O3 plus 100 nM 1,25(OH)2D3. The proportion of early apoptotic cells (Annexin V-FITC-positive and PI-negative) was 1.3% in the control; 2.8% in the cells treated with 1.0 μM As2O3; 4.0% in the cells treated with 100 nM 1,25(OH)2D3; and 0.2% in the cells treated with 1.0 μM As2O3 plus 100 nM 1,25(OH)2D3. The proportion of late apoptotic cells (Annexin V-FITC- and PI-positive) was 4.9% in the control; 30.0% in the cells treated with 1.0 μM As2O3; 8.1% in the cells treated with 100 nM 1,25(OH)2D3; and 64.3% in the cells treated with 1.0 μM As2O3 plus 100 nM 1,25(OH)2D3. The proportion of necrotic cells (Annexin V-FITC-negative and PI-positive) was 1.2% in the control; 15.8% in the cells treated with 1.0 μM As2O3; 1.5% in the cells treated with 100 nM 1,25(OH)2D3; and 33.5% in the cells treated with 1.0 μM As2O3 plus 100 nM 1,25(OH)2D3. In concordance with RT-PCR and western blot analysis, the combination treatment resulted in a more prominent apoptotic cell death compared to the single-drug treatments. In contrast, the contribution of necrosis to cell death was relatively smaller compared to apoptosis in all treatment groups.
Discussion
Previous studies have demonstrated that anti-leukemic activity of As2O3 is mainly due to its ability to induce apoptosis via various mechanisms including downregulation of Bcl-2, upregulation of caspases, and generation of reactive oxygen species (ROS) (16–18). In addition, 1,25(OH)2D3 was also found to induce apoptosis of various types of cells including colon cancer, breast cancer, prostate cancer and normal adipocytes through the activation of the apoptosis pathway (19–23). In concordance with these results, our study found that treatment with As2O3 and 1,25(OH)2D3 each inhibited the proliferation of HL-60 and K562 cells, increasing the production of pro-apoptotic Bax and caspase-3 proteins and decreasing the production of anti-apoptotic Bcl-2 protein. In addition, in the flow cytometric analysis using Annexin V-FITC and PI, the main cause of cell death induced by As2O3 and 1,25(OH)2D3 was apoptosis, which suggests an evident pro-apoptotic effect of these drugs on AML cells.
In order to overcome the resistance of As2O3, which is an active drug against APL, we aimed to evaluate the additional benefit of 1,25(OH)2D3 in combination with As2O3 on HL-60 and K562 cells. Due to the serious hypercalcemic side effect, the clinical application of 1,25(OH)2D3 single therapy to hematologic malignancies is limited in spite of its potent in vitro anti-leukemic activity (11,24). Considering this side effect, we chose a relatively low concentration of 1,25(OH)2D3, which alone could not show sufficient anti-leukemic activity. Despite its low concentration, 1,25(OH)2D3 combined with As2O3 showed an evident synergistic anti-leukemic effect on both HL-60 and K562 cells. To the best of our knowledge, this is the first study demonstrating the synergistic anti-leukemic effect of this combination treatment against AML cells. This combination treatment activated the apoptosis pathway of cells more prominently than the single treatments. Moreover, flow cytometric analysis showed that the combination treatment resulted in a more prominent apoptotic cell death compared to the single-drug treatments. The results of this study may provide evidence that low-dose 1,25(OH)2D3 may be used for improving the therapeutic efficacy of As2O3 for the treatment of patients with AML.
Since apoptosis was the dominant cause of cell death in this study, we mainly focused on the activation of the apoptosis pathway induced by 1,25(OH)2D3 and As2O3. However, As2O3 is also known to induce caspase-independent necrotic cell death via the mitochondrial death pathway (25). In this study, the effect of necrosis was relatively small when compared to apoptosis, but it was also not negligible. The proportion of necrosis in the 1,25(OH)2D3-treated cells (1.5%) did not appear to be different from that in the control group (1.2%). However, the proportion of As2O3-induced necrosis (15.8%) was substantial. In addition, 1,25(OH)2D3 profoundly increased the proportion of necrosis (33.5%) as well as apoptosis when combined with As2O3. This novel finding regarding the effect of 1,25(OH)2D3 on As2O3-induced necrosis also warrants further investigation.
It is known that both As2O3 and 1,25(OH)2D3 influence intracellular calcium homeostasis of cells, resulting in induction of apoptosis (20,23,26–28). Since these drugs share a common pathway, it is speculated that the synergistic cytotoxicity of these agents would be attributed to intracellular calcium homeostasis and their association with apoptosis induction. Although we did not investigate the calcium signaling pathway in this study, this hypothesis should be validated in subsequent studies.
In summary, low-dose 1,25(OH)2D3 in combination with As2O3 synergistically inhibited proliferation of the HL-60 and K562 cell lines. In addition, low-dose 1,25(OH)2D3 combined with As2O3 more prominently activated the apoptosis pathway than a single treatment using either 1,25(OH)2D3 or As2O3. The main cause of cell death was also apoptosis. Our results suggest that low-dose 1,25(OH)2D3 could be applied to improving the therapeutic efficacy of As2O3 against AML.