The human cancer promyelocytic leukaemia (HL-60) cell line was cultured in Roswell Park Memorial Institute media (RPMI-1640; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 100 U mL
−1 of penicillin, 100 μg mL
−1 of streptomycin, and 10% heat-inactivated FBS which was maintained in 5% CO
2 at 37 °C. The HL-60 cell line was cultured in RPMI supplemented with 10% heat-inactivated FBS at 5% CO
2 for 37 °C. The cells were sub-cultured every three days. In the cell viability assay, HL-60 cells (1.0 × 10
5 cells mL
−1) were seeded in a 96-well plate, followed by treatment of compounds at various concentrations and MTT stock solution. The quantity of formazan was measured at 540 nm by ELISA reader (Tecan Co. Ltd., Melbourne, Australia). The nuclear morphology study was carried out by seeding HL-60 cells (1.0 × 10
5 cells mL
−1) for 16 h. Subsequently, compounds were added, followed by incubation for 12 h. The nuclear morphology of the cells was evaluated using the cell-permeable DNA dye Hoechst 33342 and propidium iodide (PI). Cells with homogeneously-stained nuclei were considered viable, whereas the presence of chromatin condensation and/or fragmentation was indicative of apoptosis. The cells were placed in 24-well plates at a concentration of 1 × 10
5 cells mL
−1 (950 μL). Sixteen hours after plating, the cells were treated with various concentrations of the compounds (50 μL). After 16 h, 3 μL of Hoechst 33342 (stock 10 mg mL
−1) and PI (stock 10 mg mL
−1) were added to each well, followed by 10 min of incubation at 37 °C. The stained cells were then observed under a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera (Carsen Group, Markham, ON, Canada), in order to examine the degree of nuclear condensation. HL-60 cells (4 × 10
5 cells/mL) were treated with 150 μM of ACAN for various time points and harvested. The cells were lysed in a lysis buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO
3, 10 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 μg/mL aprotinin, and 25 μg/mL leupeptin) and kept on ice for 30 min. Antibodies against Bax, Bcl-2, caspase-9, and β-actin were purchased from Cell Signaling Technology (Bedford, MA, USA). Cell lysates were washed by centrifugation, and protein concentrations were determined using the BCA
TM protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) Aliquots of the lysates (30 μg of protein) were separated on a 12% SDS–polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (BIO-RAD) with a glycine transfer buffer (192 mM glycine, 25 mM Tris–HCl (pH 8.8), and 20% methanol (
v/v)). After the nonspecific site was blocked with 1% bovine serum albumin, the membrane was incubated with specific primary antibody (1:1000) at 4 °C overnight. The membrane was further incubated for 60 min with a peroxidase-conjugated secondary antibody (1:5000; Vector Laboratories, Burlingame, CA, USA) at room temperature.The resulting bands were visualized on X-ray film using ECL detection reagent (Amersham Biosciences, Piscataway, NJ, USA). The assay was conducted in accordance with known procedure [
32].