AA (C₃₀H₄₈O₅; molecular weight, 488.7 g/mol), dimethyl sulfoxide (DMSO), Hoechst 33258 and MTT were purchased from Sigma-Aldrich (St Louis, MO, USA). The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) reagent kit was purchased from Nanjing Key-Gen Biotech Co, Ltd (Nanjing, China). The bicinchoninic acid (BCA) Protein Assay kit, chemiluminescence reagent kit and polyvinylidene difluoride (PVDF) membranes were purchased from Pierce Biotechnology, Inc, USA. The rabbit polyclonal anti-extracellular signal-regulated kinase (ERK)1/2 (cat. no. 9102), rabbit monoclonal anti-phosphorylated (p-)ERK1/2 (cat. no. 4377), rabbit polyclonal anti-c-Jun N-terminal kinase (JNK) (cat. no. 9252), rabbit polyclonal anti-P-JNK (cat. no. 9251), rabbit polyclonal anti-p38 (cat. no. 9212), rabbit monoclonal anti-P-p38 (cat. no. 9215), rabbit polyclonal anti-Bcl-2 (cat. no. 2872), rabbit polyclonal anti-survivin (cat. no. 2803), rabbit polyclonal anti-myeloid cell leukemia 1 (Mcl-1) (cat. no. 4572), and mouse monoclonal anti-β-actin (cat. no. 3700) antibodies were provided by Cell Signaling Technology, Inc, (Danvers, MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit (cat. no. 111-035-003) and donkey anti-mouse (cat. no. 715-035-150) immunoglobulin G secondary antibodies were purchased from Jackson Immuno Research Laboratories, Inc (West Grove, PA, USA). Z-DEVD-FMK was purchased from Medical and Biological Laboratories Co, Ltd, (Nagoya, Japan). Fetal bovine serum (FBS) was obtained from Hangzhou Sijiqing Biological Engineering Materials Co, Ltd (Hangzhou, China).

The leukemia cell line HL-60 was obtained from American Type Culture Collection (Rockville, MD, USA). Human peripheral blood mononuclear cells (PBMCs) from healthy volunteers (five males and five females between 20 and 50 years old) were obtained by Ficoll-Hypaque (Lonza Ltd., Wakersville, MD, USA) density gradient centrifugation at 1,000 × g, according to the manufacturer’s instructions. All cells were cultured in RPMI 1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (Invitrogen Life Technologies) in a 5% CO₂ humidified atmosphere at 37°C. Written informed consent for blood utilization was obtained from all volunteers. The procedures were approved by the ethics committee of Tongji Medical College, Huazhong University of Science and Technology (Hubei, China).

The effects of AA on the proliferation of HL-60 cells and PBMCs were detected using an MTT assay. Briefly, the cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) until reaching the mid-logarithmic growth phase and then seeded in 96-well plates with or without AA at various concentrations (10, 20, 30, 40, 50 and 60 μmol/l) at a density of 2×10⁵ cells per well. Following incubation for a set period of time, 20 μl MTT (5 mg/ml) was added and the cells were incubated for another 3 h at 37°C. The supernatant was discarded of and 150 μl DMSO was added. The plate was gently agitated until the blue formazan crystals were fully dissolved. The absorbance (A) was measured at 490 nm using a microplate reader (Infinite F50; Tecan Spectra, Switzerland), and the cell proliferation inhibition ratio (%) was calculated using the following formula: [1-(A_{experimental sample}/A_{control sample})]×100.

The apoptotic rate was measured in HL-60 cells treated with AA at different concentrations alone or in combination with Z-DEVD-FMK. Four-color flow cytometry (FCM) was applied to detect the expression of Annexin V-FITC and the exclusion of PI. The cells positive for Annexin V-FITC and negative for PI represented the early apoptotic cells, whereas the cells positive for both markers represented the late apoptotic cells. The total apoptotic rate was the sum of the number of early and late apoptotic cells. Briefly, HL-60 cells were collected after the treatment using eppendorf tubes, washed twice with phosphate-buffered saline (PBS; Sigma-Aldrich) and resuspended in 500 μl binding buffer. A total of 5 μl Annexin V-FITC was added and the samples were maintained at room temperature for 10 min. Next, 5 μl PI was added and the cells were incubated for another 10 min in the dark. The fluorescence intensity was detected using a flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).

HL-60 cells were treated with 40 μmol/l AA for 24 h and the nuclear fragmentation was visualized using Hoechst 33258 staining. Briefly, 1×10⁵ cells were seeded in 12-well plates and incubated with AA. After 24 h, the cells were collected, washed twice with PBS and fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature before being deposited on polylysine-coated slides. After 30 min, the adherent cells were permeabilized by incubation with 0.1% Triton X-100 (Sigma-Aldrich) for 5 min at 4°C. The cells were then incubated with Hoechst 33258 for 30 min at room temperature, rinsed with PBS and mounted on coverslips using glycerol (Sigma-Aldrich). Finally, cells were visualized using an Olympus BH-2 fluorescence microscope (Olympus, Tokyo, Japan).

All of the HL-60 cells treated with AA at various concentrations for 24 h were collected. The cells were washed twice with PBS and completely lysed in a lysis buffer containing protease inhibitors (Pierce, Thermo Scientific, Waltham, MA, USA). The extracts were centrifuged at 12,000 × g for 15 min at 4°C, and the clear supernatants containing the total protein were isolated. The protein concentration was quantified using the BCA assay (Pierce, Thermo Scientific). Equal amounts of protein (40 μg per lane) were separated by 10–12% SDS-polyacrylamide gel electrophoresis, and the proteins were then transferred to PVDF membranes. The membranes were blocked in 5% non-fat milk (Sigma-Aldrich) for 2 h at room temperature and then probed with the specific primary antibodies (1:500) at 4°C overnight, and corresponding secondary antibody (1:1,000) at room temperature for 1 h. The specific protein bands were visualized using an Enhanced Chemiluminescence Detection system (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).

Each experiment was repeated three times. Values are expressed as the mean ± standard deviation and analyzed using SPSS 13.0 statistical software for Windows (SPSS, Inc., Chicago, IL, USA). The comparisons between each group were analyzed using a one-way analysis of variance and the Student-Newman-Keuls (SNK) test. P<0.05 was considered to indicate a statistically significant difference between values.

An MTT assay was used to detect the cytotoxicity of different concentrations of AA (0, 10, 20, 30, 40, 50 and 60 μmol/l) on HL-60 cells for 24 and 48 h and PBMCs for 24 h. As shown in Fig. 1, the growth of HL-60 cells was found to be inhibited by AA in a time- and dose-dependent manner. The proliferation inhibition rate of HL-60 cells significantly increased following incubation with AA at various concentrations for 24 h (P<0.05), while AA showed low toxicity to PBMCs. When the same concentration of AA was used, the proliferative rate of HL-60 cells was shown to be inhibited in a time-dependent manner (P<0.05). The IC₅₀-values of AA for HL-60 cells at 24 and 48 h were 46.67 μmol/l and 36.42 μmol/l, respectively.

Annexin V-FITC/PI double staining and flow cytometric analysis were utilized to assess the apoptotic rate of HL-60 cells treated with AA, as shown in Fig. 2. The early apoptotic rates of HL-60 cells treated with 0, 20, 40 and 60 μmol/l AA for 24 h were 4.58±2.01, 10.45±2.88 and 25.68±5.67%, respectively, which were significantly higher than that of the control group (2.53±2.06%). The late apoptotic rates were 11.20±2.91, 20.67±4.58 and 60.63±6.65%, respectively, while that of the control group was significantly lower (4.33±1.86%). To further identify whether AA-induced apoptosis in HL-60 cells is caspase-dependent, caspase inhibitor of Z-VAD-FMK was used in combination with 60 μmol/l AA. Z-DEVD-FMK was found to significantly decrease the apoptotic rate of AA-treated HL-60 cells (15.72±3.82%, P<0.05).

HL-60 cells treated with AA for 24 h were also stained with Hoechst 33258 to visualize the nuclear changes. HL-60 cell nuclei were regular in shape in the control group, whereas apparent apoptotic bodies were observed among HL-60 cells after treatment with 40 μmol/l AA. The nuclei of these apoptotic cells were condensed, and the nuclear envelopes appeared lytic (Fig. 3).

To investigate the mitochondrial apoptotic events involved in AA-induced apoptosis, changes in the levels of anti-apoptotic proteins Bcl-2, survivin and Mcl-1 were assessed by western blot analysis. AA treatment attenuated the expression levels of the anti-apoptotic proteins Bcl-2, survivin and Mcl-1 in a concentration-dependent manner in HL-60 cells (Fig. 4). These results suggested that Bcl-2, survivin and Mcl-1 have essential roles in AA-mediated induction of apoptosis.

To assess the involvement of the MAPK pathway in the mechanism underlying the cytotoxicity of AA, the levels and activation (phosphorylation) of JNK, p38 and ERK1/2 were assessed in AA-treated HL-60 cells. As shown in Fig. 5, exposure of HL-60 cells to 20, 40 and 60 μM AA resulted in a significant inhibition of phosphorylation of p38 and ERK1/2 in a dose-dependant manner, while basal activation of p38 and ERK was not altered. However, AA treatment did not JNK levels (including the phosphorylated and unphosphorylated forms).