Alpha-Santalol, a Component of Sandalwood Oil Inhibits Migration of Breast Cancer Cells by Targeting the β-catenin Pathway

Materials and Methods

Reagents. α-Santalol used in the study was isolated as described by us previously (9). Cell-culture reagents, including RPMI 1640 medium, MEM medium, FBS, and penicillin/streptomycin antibiotic mixture were purchased from Invitrogen-Life Technologies (Carlsbad, CA, USA). Six well Boyden chamber inserts were purchased from Fisher scientific (Pittsburgh, PA, USA). All the antibodies used in the study were purchased from Cell Signaling (Danvers, MA, USA). Eosin and hematoxylin were purchased from Sigma (St. Louis, MO, USA). Other chemicals used in the study were purchased in their highest purity available.

Cell lines. Cell lines of 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, PA, USA). 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 medium 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 DMSO and an equal volume of DMSO (final concentration <0.5%) was added to the controls.

Wound healing migration assay. The wound healing assay was performed by plating cells in culture dishes. In brief, a monolayer of MDA-MB 231 or MCF-7 cells were wounded by scratching with sterile pipette tips and washed with PBS. Fresh medium containing different concentrations of α-santalol or DMSO was added to the scratched monolayers for 0, 24 and 48 h. Images were taken using an inverted Nikon Bright field microscope at 100x magnification after the respective treatment period. Images were taken from three randomly selected fields and the assay was repeated twice independently.

Boyden chamber migration assay. The Boyden chamber migration assay was conducted using Transwell chambers (Costar; Cat. # 3422). Briefly 0.5 ml of MDA-MB-231 cell suspension containing 1×10⁵ cells in complete medium was mixed with either DMSO or desired concentrations of α-santalol and the suspension was added to the upper chamber. The lower compartment chamber was filled with 1.5 ml of complete medium containing the same concentrations of α-santalol or DMSO and left to incubate for 24 h. After the incubation time, the non-migratory cells that remained on the upper side of the insert membranes were removed by using a cotton swab. The cells that migrated on to the lower side of the membrane insert were fixed and stained with eosin and hematoxylin. Images of the stained cells were captured using Nikon bright field microscope at the magnification of 100×. Three randomly selected images were scored and verified by two individuals. The degree of the inhibition of migration was determined by comparing images of DMSO to images of the cells treated with α-santalol (20 μM, 40 μM). The percentage of invasive cells was expressed using untreated control cells at 100%.

Immunoblotting. Cells treated with the given concentrations of α-santalol or DMSO (control) for specific time periods were processed for immunoblotting. Lysate proteins were resolved by SDS-PAGE and transferred onto (PVDF) membrane and immunoblotting was performed as described by us previously (10). After blocking with blocking buffer (LI-COR, Lincoln, NE, USA), mixed with 0.1% Tween-20, the membrane was incubated with primary antibodies 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 antibody to correct for differences in protein loading.

Immunofluorescence assay. Cells were placed on sterile coverslips in 12-well plates and grown by overnight incubation. The next day, cells were treated with DMSO or α-santalol for 24 h. After the treatment period, the cells were rinsed with PBS and fixed with 4% formaldehyde in PBS. Cells were then incubated in blocking and permeabilization solution (10% FCS + 0.1 Triton ×100 or 0.1% Tween 100) for 1 h followed by primary antibody incubation in the blocking solution overnight. After three washes with PBS, cells were incubated with secondary antibody for 1 h. Cells were rinsed with PBS and the coverslips were mounted on glass slides using mounting media. The cells were visualized under a Nikon Bright field/fluorescence microscope at a magnification of 1000× for β-catenin localization.

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.