The ALL cell lines (CCRF-CEM, Nalm-6) were purchased from cell bank (Pasteur Institute Iran, Tehran). CCRF-CEM and Nalm-6 are derived from T and B lymphocytes, respectively. Human leukemic cell lines were grown in RPMI 1640 culture medium (GIBCO, Grand Island, NY) supplemented with 10% Fetal Bovine Serum (FBS, Gibco, Invitrogen, South America), 1% glutamine, 1% antibiotics (penicillin-streptomycin), and 1% nonessential amino acids. Cells were maintained at 37°C and 5% CO₂.

Fenretinide (4-HPR), 1α,25(OH)₂D₃ (vitamin D₃), and Bryostatin-1 (Bryo-1) were purchased from Sigma-Aldrich^®, Steibeim, Germany. All anti-cancer compounds were dissolved in 100% ethanol. Stock preparations of 4-HPR and Bryostatin-1 were stored at -20°C; however stock preparation of 1α,25(OH)₂D₃ was stored at -70°C. All reagents were diluted to appropriate final concentrations in medium (RPMI 1640). Final concentrations of ethanol never exceeded 0.01% (levels known to be without effect on the response of ALL cells to these compounds (13). Leukemic cells were washed twice in RPMI 1640 by centrifugation at 300×g for 5 min. Then, cells were suspended in medium with different concentrations of 4-HPR, 1α,25(OH)₂D₃, and Bryostatin-1. The cells were incubated for 24, 48, and 72 hr at 37°C in a humidified 5% CO₂ atmosphere. To examine the combinatory effects of 4-HPR and 1α,25(OH)₂D₃, leukemia cells were seeded at 2×10⁵ cells/ml in 24-well plastic plates and pre-treated with 0.1 or 1 nM 1α,25(OH)₂D₃ for 8, 24 or 48 hr before assays.

Cell proliferation was evaluated by the MTT (3, (4,5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl-2 (4-sulfophenyl)-2H-tetrazolium) (Sigma-Aldrich^®, Steibeim, Germany) assay (29). Briefly, 5×10⁴ cells were plated in a total volume of 100 µl in 96-well plates (FALCON, USA) and suspended in medium with different concentration of 4-HPR, 1α,25(OH)₂D₃, and Bryostatin-1(Bryo-1). MTT was dissolved in absolute ethanol. Following 24, 48, and 72 hr incubation, 0.01 ml of MTT solution (at a final concentration 0.5 mg/ml) was added per well. The plates were then incubated for additional 3 hr. Then 100 µl of stop solution (isopropanol containing 0.04% HCl) was added per well. Immediately after solving the blue formazon crystals, the absorbance of samples was read using a 96-well plate reader (Anthos 2020) at 570 and 630 nm wave lengths. Results reported in this article are the mean±S.E.M. of three independently performed experiments, and each concentration was tested in eight wells per experiment. The results were considered to be significant when the p-value was <0.05, and highly significant when the p-value was <0.01 or <0.001.

The DNA content during cell cycle steps were evaluated with flow cytometry. In brief, 5×10⁶ cells were treated with drugs at a specific concentration. After 24 hr, the cell pellets were washed and resuspended in 2 ml of 1% paraformaldehyde in PBS and incubated for 15 min at 4°C. Then, cells were centrifuged and 1 ml of cold perm buffer III (BD. Co, USA) solution was added; cells were incubated for 30 min at 4°C and then were washed twice in PBS. Next, 500 λ of PI (Sigma-Aldrich^®, Steinbeim, Germany) staining buffer (50 µg/ml PI, 10 µg/ml RNase in PBS) was added and incubated for 1 hr at room temperature in the dark. After DNA staining by Propidium Iodide (PI), samples were evaluated by a flow cytometer using Partec FloMax software (Version 2.3) (29).

In this study, 1×10⁶ cells/ml suspension of ALL cell lines was induced for apoptosis by addition of several concentrations of drugs. 1x10⁶ cells/ml suspension of non-induced leukemic cells was established as a negative control. Both control and experimental leukemic cell samples were incubated for 24 and 48 hr in a 37°C, 5% CO₂ incubator. Following incubation, cells were washed twice with DPBS (Dulbecco's Phosphate Buffered Saline). After that, the cells at concentration of about 1×10⁶ cells/ml were resuspended in 1X binding buffer (100 mM HEPES/NaOH, pH 7.5 containing 1.4 M NaCl and 25 mM CaCl2). Five hundred λ of the apoptotic cell suspension was added to a plastic 12 x 75 mm test tube, and 500 λ of the non-induced cell suspension was added to a second plastic tube. Next, 5 λ of AnnexinV-FITC (Sigma-Aldrich^®, Steibeim, Germany) and 10 λ of Propidium Iodide (PI) (Sigma-Aldrich^®, Steibeim, Germany) were added to each cell suspension. Then the tubes were incubated at room temperature for exactly 10 min and protected from light. Finally, fluorescence of the cells was immediately determined by a flow cytometer (29).

In order to adjust the flow cytometer for evaluating the apoptosis, a positive and a negative control sample was used. As a positive control, apoptosis was induced in a 1×10⁶ cells/ml suspension of leukemic cells by addition of 1 µg/ml Staurosporine (Sigma-Aldrich^®, Steibeim, Germany). Flow cytometry analysis was performed using Partec FloMax software (Version 2.3).

The ALL cell lines were analyzed for phenotypic evidence of differentiation by examining the expression of cell surface antigens as described previously (30, 31). Briefly, CCRF-CEM and Nalm-6 cells were washed with PBS supplemented with 1% FBS and stained with the following antibodies for 30 min at 4°C: R-phycoerythrin (RPE)-conjugated mouse anti-human CD19 IgG1, R-phycoerythrin (RPE)-conjugated mouse anti-human CD38 IgG1, or fuorescein isothiocyanate (FITC)-conjugated mouse anti-human CD7 IgG2b (DakoCytomation, DAKO, Denmark). Specifically, CCRF-CEM cells were stained with FITC-conjugated mouse anti-human CD7 IgG2b and Nalm-6 cells were stained with RPE-conjugated mouse anti-human CD19 IgG1 or RPE-conjugated mouse anti-human CD38 IgG1. Cells were then washed to remove unbound antibody, resuspended in 0.5 ml PBS containing 1% FBS for immediate analysis with a minimum acquisition of 2×10⁴ events. Samples were run on a Partec FloMax flow cytometer. Results are presented as the relative mean fuorescence after subtracting the isotype control for each sample compared to the untreated media controls.

Cell lysates (from 6×10⁶ cells) were assayed for protein concentration with the BCA Protein Assay Reagent kit (Thermo scientific, U.S.A). Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis was performed as previously described (32, 33). Proteins were resolved on a 12% SDS polyacrylamide gel, transferred to a PVDF membrane (Roche, Germany). After transferring to PVDF membrane and blocking the non-specific binding sites with 5% skim milk, the membrane was incubated with the human reactive monoclonal anti-caspase-3 (abcam, Mediqip, United States) for 2 hr followed by incubation with the secondary rabbit anti-mouse horseradish peroxidase-labeled anti-body (1:1000) for one hr. Bound antibody was detected with the ECL Western Blotting Detection System (GE healthcare, U.K). The chemiluminescence of the membranes was detected by exposure to X-ray film (Kodak, Rochester, NY). Hybridization with the house keeping protein β-Actin was used as a control for equal loading and protein quality.

Statistical analyses were carried out using SPSS software (PASW 18, an IBM Company, Iran). Data are expressed as the mean±S.E.M. and comparison between the controls and each treatment group was performed using a two-tailed, paired student's t-test. The results were considered to be significant when the p-value was <0.05, and highly significant when the p-value was <0.01 or <0.001.

Recently, we have shown that 4-HPR can prevent the growth and proliferation of an AML cell line (NB-4) (34). In the present study, to determine the growth inhibitory effect of 4-HPR on two acute lymphoblastic leukemia cell lines (CCRF-CEM, and Nalm-6), both cell types were exposed to increasing concentrations of 4-HPR (1, 2.5, 5, 7.5, and 10 µM) and cell viability was determined by MTT assay. Eight samples were tested for each drug concentration and each experiment was repeated three times before MTT analysis. Results are represented in Figure 1. 4-HPR inhibits the growth of ALL cell lines in a concentration-dependent manner (Figure 1A) and the most inhibitory effects were observed in Nalm-6 cells at low micromolar (µM) range after 72 hr. Overall, the results indicate that Nalm-6 cells relative to CCRF-CEM cells are more sensitive to fenretinide treatment.

1α,25(OH)₂D₃ was another compound investigated in the present study. Based on previous studies (13, 35), concentrations in the nanomolar range were chosen to treat the cell lines (0.1, 10, and 100 nM). In Figure 1B, effect of 1α,25(OH)₂D₃ on growth of CCRF-CEM and Nalm-6 cell lines is demonstrated. The maximum concentration used in this study (100 nM) revealed the highest inhibitory effects on growth of both leukemic cell lines. In Nalm-6 cells, 1α,25(OH)₂D₃ induced 73% inhibition at 100 nM, 48 hr post treatment. Nalm-6 cell line was more sensitive to 1α,25(OH)₂D₃ treatment relative to CCRF-CEM.

The third anti-cancer drug chosen to determine its growth inhibitory effects on ALL cells was Bryostatin-1. Although the concentrations of Bryostatin-1 used in this study were in the low nanomolar (nM) range, the results indicate a significant decrease in the survival of two leukemic cell lines. For instance, as shown in the Figure 1C, Bryostatin-1, at concentration 10 nM, inhibits proliferation of CCRF-CEM and Nalm-6 cells by 63.03% and 60.25%, respectively (48 hr post treatment).

To show the effective dose of each anti-cancer drug that inhibited 50% growth (ED₅₀) of ALL cells after 24, 48, and 72 hr treatment, ED₅₀ was determined by plotting the inhibition of cell proliferation in the presence of increasing concentration of 4-HPR (1-10 µM), VD₃ (0.1-100 nM), and Bryostatin-1 (0.1-10 nM) (Table 1). Comparing the ED₅₀s among the three drugs, 4-HPR potently inhibited the growth of CCRF-CEM and Nalm-6 cells (ED₅₀ ranging from 4.6 to 7.2 µM); however, it is clear that Bryostatin-1 has the lowest and clinically acceptable ED₅₀ in lymphoblastic leukemia cell lines (24, 28).

Because previous studies demonstrated the cooperative action of retinoic acid derivatives and 1α,25(OH)₂D₃ in various cancer cells (36, 37), we next examined the synergistic effects of 4-HPR and 1α,25(OH)₂D₃ on proliferation inhibition in acute lymphoblastic leukemia cell lines (CCRF-CEM and Nalm-6) using MTT assay. Figures 1D and 1E respectively show that combination of low concentrations of 4-HPR (1 and 2.5 µM) and 1α,25(OH)₂D₃ (0.1 and 1 nM) can significantly reduce viability of ALL cells (up to 80%).

To investigate the mechanisms by which 4-HPR, 1α,25(OH)₂D₃, and Bryostatin-1 inhibited the growth of ALL cell lines, the cell cycle distribution was analyzed after exposure of these cells to the anti-cancer drugs. As shown in Table 2, the untreated CCRF-CEM cells (T-ALL) were mainly in G2/M phase (51.61%), and with the treatment of 5 µM 4-HPR and 100 nM 1α,25(OH)₂D₃ for 24 hr, the percentage of S phase cells were significantly decreased and those of G2/M phase cells were correspondingly increased (65.6% and 63.22% respectively).

In contrast, treatment of CCRF-CEM cells with Bryostatin-1 (10 nM, 24 hr) significantly decreased the fraction of G2/M cells (26.83%) and increased the fraction of G0/G1 cells (46.8% compare to control 29.54%). Table 2 shows that untreated Nalm-6 cells (B-ALL) were mostly in G0/G1 (65.35%). When treated with 4-HPR (5 µM), 1α,25(OH)₂D₃ (100 nM), and Bryostatin-1 (10 nM), B-ALL cells were markedly accumulated in G0/G1 phase (86.42%, 76.1%, and 89.6%, respectively). Figure 2 is a representative flow cytometric chart of effects of anti-cancer drugs on cell cycle distribution in acute lymphoblastic leukemia cells.

Next we examined the involvement of apoptotic cell death in reduced survival of CCRF-CEM and Nalm-6 cells in response to three anti-cancer drugs. For this purpose, Annexin V staining and flow cytometric analysis was utilized. The apoptosis sensitivities of the investigated cell lines were looked at in three groups of 4-HPR-treated, 1α,25(OH)₂D₃-treated, and Bryostatin-1-treated cells (Tables 3A, 3B, and 3C). In Table 3A, CCRF-CEM and Nalm-6 cells treated by 4-HPR demonstrate an increase in apoptosis as the concentrations change. Table 3B shows that at higher concentrations of 1α,25(OH)₂D₃, CCRF-CEM cells were more responsive than Nalm-6 cells in 48 hr treatment group. As results in Table 3C show, exposure of CCRF-CEM and Nalm-6 cells to Bryostatin-1 (24 hr and 48 hr) increased the population of cells being positive for annexin V (4.5-12.11%).

Next we evaluated the synergistic effects of 4-HPR and 1α,25(OH)₂D₃ on apoptosis induction in ALL cell lines. Table 3D shows that combination of 4-HPR and 1α,25(OH)₂D₃ increase the fraction of ALL cells undergoing apoptosis 24 and 48 hr post treatment when compared to individual treatments. Figure 3 is a representative flow cytometric chart demonstrating induction of apoptosis by anti-cancer drugs in acute lymphoblastic leukemia cells.

In recent years, many studies have been reported on the AML differentiation and differentiation induction by anti-cancer drugs (30, 31, 38). However, very few studies on differentiation induction in ALL cells are available (39–41). Several investigations have shown that B-cell differentiation is correlated with an upregulation of both CD19 and CD38 expressions (42–44). In this study, we examined the potential capacity of 4-HPR, 1α,25(OH)₂D₃, and Bryostatin-1 for differentiation induction in B-ALL (Nalm-6) cells. Figures 4A and 4B show that treatments by three anti-cancer drugs strongly induce up-regulation of CD19 and CD38 in Nalm-6 cells.

To examine differentiation induction in CCRF-CEM (T-ALL) cells, we evaluated the presence of CD7 expression on these cells before and after treatment with three anti cancer compounds. We showed that all three anti-cancer drugs significantly increased CD7 expression in CCRF-CEM cell line (Figure 4C). A representative flow cytometric chart of these experiments is shown in Figure 5.

Since all three anti-cancer compounds (fenretinide, 1α,25(OH)₂D₃, and Bryostatin-1) induce apoptotic cell death in CCRF-CEM and Nalm-6 cell lines, we evaluated the mechanism by which these anti-cancer drugs act. Activation of caspase-3, a key enzyme involved in apoptotic cell death, was examined in ALL cell lines treated by drugs for 24 hr using Western blotting. Caspase-3 activity was observed in ALL cells when compared to control cells (ALL cells without treatment) suggesting that caspase-3 is activated in ALL cells treated with 4-HPR, 1α,25(OH)₂D₃, and Bryostatin-1 (Figure 6). We monitored uniform expression of β-Actin as a loading control.