Human acute lymphoid leukemia cell line CCRF-CEM and Molt-4 were kindly presented by Dr Y.Y. Lee (Hanyang University, Seoul, Korea). The cells were cultured in tissue flasks or plates in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco-BRL, Gaithersburg, MD, USA), 100 U/ml penicillin, 100 µg/ml streptomycin and incubated at 37°C in a humidified atmosphere with 5% CO₂.

KML001 was obtained from Komipharm International (Siheung-Si, Gyeonggi-Do, Korea). As₂O₃ was purchased from Sigma-Aldrich (St. Louis, MO, USA). Doxercalciferol was purchased from Selleck Chemicals (Houston, TX, USA). Antibodies of Cdk1, p-Cdk1, cyclin B, p-p53, p-p21, Bcl-2, Bax, Bcl-xL were from Abcam (Cambridge, UK).

Cell viability was determined by Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Gaithersburg, MD, USA). Cells (5×10³ cells/well) were seeded in 96-well microtiter plates (Falcon) and then incubated at 37°C for 48 h. After adding 10 µl of the CCK-8 solution to each well of the plate, the plate was incubated for ≥1 h in the incubator. Finally, absorbance at 450 nm was measured by Multiskan Spectrum microplate reader (Thermo Labsystems, USA).

Cells were lysed with lysis buffer [25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS with Halt™ Protease, Phosphatase Inhibitor Cocktail and Benzonase^® Nuclease] on ice for 20 min. Briefly, protein samples were resolved in SDS-polyacrylamide gel, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and then probed with antibodies overnight. Finally, the blots were developed using the Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, USA).

Cells were fixed with 66% ethanol for >2 h at 4°C and then stained with 50 µg/ml of propidium iodide (PI) containing 550 U/ml of RNaseA (ab139418 PI Flow Cytometry kit for cell cycle analysis (Abcam, MA, USA). The DNA content of the cells was gated and analyzed using a FACSCanto II (Becton-Dickinson, San Jose, CA, USA) equipped with an FACSDiva software 8.0.1 (Becton-Dickinson).

Apoptosis was evaluated by FITC Annexin V apoptosis detection kit according to the manufacturer's instructions. Cells were washed twice with cold PBS, then resuspended in the 1X binding buffer, containing 0.01 M HEPES/NaOH, 0.14 M NaCl and 2.5 mM CaCl₂, pH 7.4. Next, a total of 100 µl of the mixtures was transferred to a 5-ml culture tube and then incubated with 5 µl Annexin V-FITC and 5 µl propidium iodide for 15 min at room temperature in the dark. Subsequently, 400 µl of 1X binding buffer was added to each tube and then cells were immediately analyzed by flow cytometry. In analysis process, Annexin V-positive or PI-negative cells were considered as early apoptotic, both Annexin V and PI-positive cells were considered as late apoptotic, and Annexin V-negative but PI-positive cells were considered as necrotic.

Cells treated with KML001, doxercalciferol or medium (control) for 48 h were washed three times with the PBS and then incubated with 4 µM Fluo-4 AM (Thermo Fisher Scientific) and 0.1% Pluronic F-127 (Thermo Fisher Scientific) in PBS for 60 min at 37°C. After removing the medium in time, each well was washed with PBS and 200 µl PBS was used to cover all cells in the trough. Finally, we used laser confocal scanning microscopy (LCSM) to measure the fluorescence intensity (FI) of the cells at an excitation wavelength of 488 nm.

The combined effects of KML001 and doxercalciferol were analyzed by CalcuSyn software (Biosoft Ferguson, MO, USA) using the Chou-Talalay method (19). The combination index (CI) values of <1, 1 and >1 represent synergy, additive effect, and antagonism, respectively. All experiments have been repeated at least 3 times. Statistical significance was determined by using Student's t-test. P-value <0.05 was considered statistically significant.

The current experiments were initiated by treating CCRF-CEM and Molt-4 cells, in in vitro models of human ALL, for 48 h with two types of arsenic compounds, arsenic trioxide and KML001. Results showed that KML001 inhibited cell proliferation of the cell lines at a fairly lower concentration (>100-fold) than As₂O₃ (Fig. 1).

To further investigate the mechanism of action of KML001, western blot analysis was performed for apoptosis-related proteins in CCRF-CEM and Molt-4 cells (Fig. 2A). In both cell lines, decreased Bcl-xL and increased Bax were observed after 48-h treatment of KML001. Expression of anti-apoptotic protein Bcl-2 decreased markedly in CCRF-CEM cell line while its expression increased a little in the Molt-4 cell line.

Cdk1/cyclin B1 complex, the critical regulator of G2/M checkpoint, plays important roles in mitosis and mitotic catastrophe (20,21). As shown in Fig. 2B, the expression of Cdk1 kept decreasing after treatment of KML001 while cyclin B1 expression did not show any change. Lack of Cdk1 may cause cell arrest at G2/M phase. To confirm this point, we conducted cell cycle analysis by propidium iodide staining of the treated cells followed by flow cytometry (Fig. 2C). As shown by the G2/M ratios in both cell lines (Fig. 2D), KML001 did induce cell cycle arrest both in CCRF-CEM and Molt-4 cells. Previous studies suggest that p53 and p21 were involved in both cell cycle arrest and cell apoptosis (22,23). Here, we found p53/p21^WAF1 pathway was involved in KML001-induced G2/M phase arrest. Fig. 2B indicates that p-p21 expression increased in a time-dependent manner in CCRF-CCEM and Molt-4 cells after KML001 treatment. In addition, increased p53 phosphorylation was observed in CCRF-CEM cells but not in Molt-4 cells which are p53-deficient.

We also investigated whether KML001 and doxercalciferol could cause synergistic effects in terms of reduced cell viability in ALL cell lines. CCRF-CEM and Molt-4 cell lines were incubated for 48 h with one drug alone or with a combination treatment of KML001 and doxercalciferol at a constant ratio of 1:312.5 or 1:625 (KML001:doxercalciferol). CI and cell viability were then calculated. As shown in Table I, in the two cell lines, the combined treatments were synergistic, as indicated by CI <1.

Also, we observed a significant increase of late apoptotic cells (double-positive for Annexin V and PI) after combination treatment compared with single treatment of KML001 or doxercalciferol (Fig. 3).

Western blot analysis and flow cytometry were performed to know whether doxercalciferol induces cell cycle arrest in ALL cells. As shown in Fig. 4A, expression of Cdk1 and cyclin B decreased in a time-dependent manner after treatment, which indicates that doxercalciferol may also lead to G2/M phase arrest of these two cell lines. Flow cytometry analysis indicates that doxercalciferol was able to induce cell cycle arrest at G2/M phase in CCRF-CEM and Molt-4 cells (Fig. 4B and C).

To further identify mechanisms underlying this synergy, we also tried to understand mechanisms of action of doxercalciferol in ALL models in vitro. The pro-apoptotic protein Bax and anti-apoptotic protein Bcl-xL did not change as we expected (Fig. 5A). Considering the late apoptotic cells observed in flow cytometry results, doxercalciferol may cause cell apoptosis without the involvement of Bcl-2 family pathway.

In addition, we tried to understand mechanisms of doxercalciferol-induced G2/M phase arrest by studying intracellular signaling pathway through western blot analyses. However, doxercalciferol neither induce p53 activation nor p21 activation in the two cell lines (Fig. 5B).

Finally, to investigate whether doxercalciferol-induced apoptosis is associated with Ca²⁺, the effect of doxercalciferol on intracellular calcium ion concentration in ALL cells was observed by using LSCM after staining with the fluorescent probe, Fluo-4/AM. As shown in Fig. 5C-L, the depth of the green signal, which indirectly reflects intracellular Ca²⁺ concentration, increased as the time increased after treatment of doxercalciferol while there was relatively less change observed in groups treated with KML001.