Cell culture
Cell lines were derived from the SKOV3 human epithelial ovary cancer cell line (ATCC, USA). Cells were grown in high glucose (4.5g/l) Dulbecco’s Modified Eagle Medium containing 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100mg/ml streptomycin. Cells were cultured at 37°C in a humidified incubator with 5% CO2 and 95% air.
Cell viability assay
Cell viability was determined MTS assay kit (CellTiter 96 Aqueous One Solution, Promega). Curcumin was dissolved in dimethyl sulfoxide, stored at −20°C, and diluted to the desired concentration immediately before the experiments. SKOV3 cells were seeded and allowed to adhere overnight. Then, cells were incubated in FBS-free media containing various concentrations of curcumin (0, 10, 20, 30, 40, and 50 μM). MTS reagent was then added to each of the wells and were incubated at 37°C for various times (6, 24, 48, and 72 hours). The optical density was measured at 490nm with a microplate spectrophotometer.
Western blot analysis
Fascin expression was quantified by western blot analysis. SKOV3 cells were incubated in FBS-free media containing 0 and 10 μM curcumin for 6 hours and washed with PBS. Cells were collected after 6, 12, and 24 hours and lysed with a lysis buffer solution. The cell lysate was centrifuged at 13,000 rpm for 30 min at 4°C, the supernatant was separated, and the proteins were quantified by the bicinchoninic acid protein assay (Sigma, St. Louis, MO, USA). Protein samples were separated on 10% SDS-polyacrylamide electrophoresis gels and then transferred to nylon membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 for 1 hour at room temperature. After incubation with a 1:1000 dilution of mouse monoclonal anti-Fascin (Santa Cruz, CA, USA) at 4°C overnight, peroxidase-conjugated secondary antibody was added for 1 hour at room temperature. The fluorescent signal was detected using an enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, England). Band densities were calculated using image J software, version 1.46r, computer-assisted image analyzer (National institutes of Health, USA).
Sandwich ELISA
To determine the effect of curcumin on the upstream pathway of fascin expression, the signal transducer and activator of transcription 3 (STAT3) was analyzed by sandwich-ELISA. STAT3 is known as a transcription factor related to a master regulator of ovary cancer cells that is essential for maintaining a tumor’s initiating capacity and ability to invade the normal tissue. After SKOV3 cells were incubated in FBS-free media containing 10μM curcumin. Cells were collected after 6, 12, and 24 hours and lysed with an ice-cold lysis buffer solution. The concentrations and phosphorylation status of the transcription factor STAT3 in the samples were determined using a sandwich-ELISA kit (PathScan® Phospho-Stat3 (Tyr705) Sandwich-ELISA Antibody Pair #7146; Cell Signaling Technology Inc., Danvers, MA, USA). After coating the microplate wells, 100μL of the respective lysates were added to each well and incubated at 37°C for 2 hours before the wells were washed, and a detection antibody was added for 1 hour. Then, 100 μL of a secondary polyclonal antibody, conjugated to horseradish peroxidase, was added to each well. The plate was then incubated for 30 minutes at room temperature. Finally, a 200μL of substrate solution (Tetramethylbenzidine) was incubated in each well for 10 minutes at 37°C. The reaction was terminated by adding 100μL of stop solution (2N sulfuric acid). The color in the wells changed from blue to yellow. The optical density of each well was measured using a microplate reader (VERSA max, Molecular Device Inc., CA, USA) set to 450 nm.
Attachment assay
Ninety-six well plates were incubated at 4°C overnight with laminin at 10μg/ml. The unbound sites were blocked with 0.1% BSA for 1 hour. SKOV3 cells, which were temporarily treated with 10 and 20 μM curcumin for 6 hours, were seeded at a density of 5 × 104 cells/well on laminin or BSA-coated plates. The cells were allowed to adhere for 6, 12, and 24 hours at 37°C in a humidified incubator supplied with 5% CO2. After washing with PBS, the remaining cells were fixed with 4% paraformaldehyde for 10 min and then stained with 5% crystal violet for 20 min at room temperature. Cells were solubilized with 1% SDS and the absorbance of each well was measured using a microplate reader (VERSA max, Molecular Device Inc., CA, USA) set to 595nm.
Migration assay
SKOV3 cells were plated in 6-well dishes at a density of 3 × 105 cells/well and were allowed to attach and reach 80% subconfluency. Thereafter, they were incubated with a starvation medium containing 10 and 20μM curcumin for 6 hours. A scratch was formed through the cell monolayer using a yellow pipet tip in FBS-free media. Cells were photographed using an Axioplan-2 epifluorescence microscope (Carl Zeiss Vision GmbH, Munchen, Germany) after 6, 12, and 24 hours. The images were viewed on a computer monitor using a Zeiss Plan-Apochromat 40x objective (Carl Zeiss Vision GmbH, Munchen, Germany). Areas of the scratch were counted with image J, version 1.46r, computer-assisted image analyzer (National institutes of Health, USA).
Invasion assay
Cell invasion was evaluated by a Matrigel-coated modified Boyden chamber (BiocoatTM MatrigelTM Invasion Chamber; Becton Dickinson GmbH, Heidelberg, Germany). Approximately 2.5x104 control and SKOV3 cells treated with 10 and 20 μM curcumin for 6 hours were seeded into the upper well of the chamber containing 500μL serum free culture medium. A 500μL culture medium with 10% FBS was added to the bottom of the well. After 6, 12, and 24 hours, non-invading cells in the top chamber were removed gently with a cotton swab, cells on the bottom of the chamber were fixed with 4% paraformaldehyde for 15 minutes at room temperature, and then stained with 0.5% crystal violet at room temperature for 10 minutes. After the crystal violet was rinsed with distilled water, cells were photographed using an Axioplan-2 epifluorescence microscope (Carl Zeiss Vision GmbH, Munchen, Germany). The images were viewed on a computer monitor using a Zeiss Plan-Apochromat 100x objective (Carl Zeiss Vision GmbH, Munchen, Germany).
Immunofluorescence
To determine the morphological changes of ovarian cancer cells by curcumin, immunofluorescence was performed. SKOV3 cells were seeded at a density of 5,000 cells/well in a 96-well plate and allowed to adhere overnight. After temporary exposure to 0 (control) and 10μM curcumin for 6 hours, the cells were washed three times with PBS. After 6 and 24 hours, cells were fixed in 4% paraformaldehyde (PFA) for 10 minutes and permeabilized for 5 minutes with 0.5% Triton X-100 in PBS at room temperature. Nonspecific binding was blocked with 1% BSA in PBS for 1 hour at room temperature. Subsequently, cells were incubated with an anti-Fascin 1 monoclonal antibody (1:100, sc-46675, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hour. After washing, cells were then incubated with Alexa Fluor 594-conjugated goat anti–mouse IgG secondary antibodies (Molecular Probes, Eugene, OR, USA) for 30 minutes at room temperature. The images were viewed on a computer monitor using a Zeiss Plan-Apochromat 40x objective (Carl Zeiss Vision GmbH, Munchen, Germany).
Statistical analysis
All statistical comparisons were calculated using SPSS 22.0 software (SPSS, Inc., an IBM Company, Chicago, IL, USA). Data were expressed as mean ± standard error of the mean. Repeated measure ANOVA was used to compare groups. Null hypotheses of no difference were rejected if p-values were less than 0.05.