Survivin Down-regulation by α-Santalol Is Not Mediated Through PI3K–AKT Pathway in Human Breast Cancer Cells

Materials and Methods

Materials. α-Santalol used in the study was isolated as described by us previously (14). Cell-culture reagents, including RPMI-1640 medium, minimal essential medium (MEM), fetal bovine serum (FBS), and penicillin/streptomycin antibiotic mixture were purchased from Invitrogen-Life Technologies (Carlsbad, CA, USA). ELISA kit for survivin activity was purchased from Cell Signaling (Danvers, MA, USA) and caspase-3 activity ELISA kit was purchased from Invitrogen (Camarillo, CA, USA). All the antibodies used in the study were purchased from Cell Signaling.

Cell lines. Cell lines MDA-MB-231 and MCF-7, which were previously authenticated by Research Animal Diagnostic Laboratory (University of Missouri, Columbia, MO, USA) to test for interspecies contamination and alleles for short tandem repeats identifiable in the ATCC database, were a generous gift from Dr. Shivendra V. Singh (University of Pittsburgh Cancer Institute, Pittsburgh). MDA-MB-231 cells were cultured in RPMI Nutrient Mixture supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin antibiotic mixture. The MCF-7 cells were maintained in MEM supplemented with sodium pyruvate (1 mM/l), non-essential amino acids (1 mM/l), 10% (v/v) FBS, and 1% penicillin/streptomycin antibiotic mixtures. Each cell line was maintained in an atmosphere of 95% air and 5% CO₂ at 37°C. A stock solution of α-santalol was prepared in dimethyl sulfoxide (DMSO) and an equal volume of DMSO (final concentration <0.5%) was added to the controls.

Cell viability assay. The effect of α-santalol on cell viability in MDA-MB-231 cells was determined using Trypan blue dye exclusion assay as described by us previously (12). Briefly, cells (20×10⁵) were plated in 12 well plates, followed by overnight incubation, cells were treated with DMSO-control or α-santalol (20, 40 μM) for 24 hours. After the treatment time period, cells were collected and analyzed for cell viability using Trypan blue dye exclusion assay.

Survivin assay. Survivin sandwich ELISA kit was used to detect the endogenous levels of total survivin protein. Briefly, cells (5×10⁵) were plated in tissue culture dishes, allowed to attach by overnight incubation and exposed to DMSO-control or α-santalol (20, 40 μM) over 6 and 9 hours. Cells were collected and lysed using 1x cell lysis buffer (provided in the kit) supplemented with 1 mM phenylmethylsulfonyl fluoride. Survivin ELISA was carried out according to the manufacturer's recommendations. Briefly, cell lysates (60 μg) from control and treated groups were diluted with appropriate volume of sample diluent to obtain a total volume of 100 μl of the diluted lysates. The diluted lysates were added to microwells, which were then sealed and incubated (overnight, 4°C). Following incubation, contents of the wells were discarded by gentle tapping and washed (4 times) with 1x wash buffer and incubated with detection antibody (100 μl, 1 h, 37°C). After removal of the detection antibody solution, the wells were washed again and incubated with horse radish peroxidase (anti-rabbit antibody (100 μl, 30 min, 37°C). After aspiration of the secondary antibody, substrate solution (100 μl) was added and plates were again incubated (10 min, 37°C). The reaction was stopped by adding stop solution (100 μl) and the yellow color developed, an indication of survivin present in the samples, was read using a microplate reader (450 nm) within 30 min after adding the stop solution.

Immunoblotting. Cells treated with 20 or 40 μM of α-santalol or DMSO (control) for 6 or 9 h were processed for immunoblotting. Lysate proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membrane and immunoblotting was performed as described by us previously (12). After blocking with blocking buffer (LI-COR, Lincoln, NE, USA), mixed with 0.1% Tween-20, the membrane was incubated with primary antibodies (survivin, pAKT, total AKT and actin) overnight at 4°C. Following incubation with appropriate secondary antibody, the immunoreactive bands were visualized using LI-COR-Odyssey infra-red scanner (LI-COR). The blots were re-probed with anti-actin to correct for differences in protein loading.

Caspase-3 activity assay. The effect of α-santalol on caspase-3 activity in MDA-MB-231 cells was determined using a commercially available caspase-3 (active) ELISA kit, as described by us previously (12). Active caspase-3 (ng/mg total protein of cell lysate) was determined and results were expressed as the fold of caspase-3 activity in α-santalol-treated cells relative to that of the DMSO-treated control. ELISA assay for caspase-3 activity was carried out according to the manufacturer's instructions.

Pharmacological inhibition of AKT pathway. Inhibition of AKT pathway was carried out by employing LY294002 (commercially available pharmacological inhibitor of AKT pathway). Briefly, MDA-MB-231 cells (5×10⁵) were plated in tissue culture dishes, allowed to attach by overnight incubation. Next day cells were pretreated with 20 μM of LY294002 for 2 h and co-treated with DMSO (control) or 40 μM of α-santalol for 24 h. Cells were then collected and processed for survivin assay and caspase-3 activity assay as described earlier.

Statistical analysis. Each experiment was carried out at least twice with triplicate measurements for quantitative comparisons. Statistical significance of difference in measured variables between control and treated groups was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. Differences were considered significant at p<0.05.