Cell lines and culture conditions
The cisplatin-sensitive A2780 ovarian adenocarcinoma cell line and its daughter line, A2780cis, were obtained from the European Collection of Cell Cultures (Salisbury, UK). Cells were cultured in RPMI with 10%FBS, 1% penicillin/streptomycin and 1% L-glutamine at 37°C in 5% CO2. A2780cis cells were maintained in media with 1 μM cisplatin. For all assays, cells were grown to 70-80% confluence and harvested following trypsinization. Analysis of cell morphology was done at 20× magnification using a Zeiss Axiovert 25 Phase Contrast Inverted Microscope. Digital images were captured using a Canon Power Shot G10 equipped with a Carl Zeiss 426126 lens.
Human ovarian tumours
Consent for tumour banking was obtained and the study was approved by the Research Ethics Boards at both Kingston General Hospital and The Ottawa Hospital. Tumour samples were obtained from the Division of Gynecologic Oncology Ovarian Tissue Bank and the Ontario Tumour Bank. All tumours were chemonaïve at collection. Seventeen tumours were classified as chemosensitive (progression-free interval of greater than 18 months) and eleven as chemoresistant (progression-free interval of less than 8 months) using available follow-up clinical data. Histological assessment of samples confirmed that each sample contained > 70% tumour cells.
Gene expression profiling and analysis
Total RNA was extracted from the cell lines and tumours using the Qiagen miRNeasy Mini kit (Toronto, Canada). RNA quality was assessed to have an RNA integrity number of at least eight using an Agilent 2100 Bioanalyzer (Mississauga, Canada). For cell lines, total RNA was labeled with Cyanine-3 dye using a Quick Amp Labeling Kit (Agilent, Mississauga, Canada) and hybridized to Agilent Whole Human Genome (4 × 44 K) Microarrays (Mississauga, Canada) for 17 hours in a rotating SciGene model 700 oven (Sunnyvale, USA). Arrays were scanned using an Agilent Technologies DNA Microarray Scanner and data were feature extracted using Feature Extraction Software 10.5.1.1 (Agilent) and statistically analyzed using default settings on GeneSpring GX 11.0.1 software (Agilent).
Expression profiles from the tumor RNA were obtained using Affymetrix GeneChip Human Genome U133 plus 2.0 arrays. Raw data were imported into GeneSpring GX 11.0.1 and analyzed. Unsupervised hierarchical clustering of the tumour samples was completed using the self-organizing maps algorithm in the GeneSpring GX 11.0.1 package.
qRT-PCR Taqman™ arrays
Snail and Slug expression levels were analyzed using Taqman™ assays (Applied Biosystems, Streetsville, Canada, item Hs00195591_m1 and item Hs00950344_m1) and the SuperScript III First-Strand Synthesis SuperMix kit for qRT-PCR (Invitrogen, Burlington, Canada). PCR conditions were 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, 60°C for 1 minute. GAPDH was used as an internal control. As a measure of relative change in expression between the parental and resistant samples, ΔΔCt values were calculated and converted to approximate fold change values (2-ΔΔCt) .
Cell proliferation assay
Cells were plated in 24-well plates at 5 × 104 cells/well. Cells were harvested and counted using a haemocytometer after 24, 48 and 72 hours. Average cell counts were used to produce growth curves, from which cell doubling time was calculated.
Wound healing assay
Cell migration was assessed using wound-healing assays. Cells were grown in a confluent monolayer in a 60 mm plate. A wound was inflicted in the cell layer by scratching the plate with a sterile pipette tip. Plates were rinsed gently with media twice prior to incubation to remove non-adherent cells. Digital images of the wound were obtained at times 0 hours, 12 hours and 36 hours at 10× magnification. Effects of proliferation were controlled for by using a reduced serum medium (3%FBS) and monitored via cell count.
Boyden chamber migration and invasion assays
Cells were serum starved for 24 hours prior to use. Media with 10% FBS was added to the wells of a 24-well plate. BD Falcon™ Cell Culture Inserts (BD Biosciences, Mississauga, Canada) were placed in each well. 2 × 103 cells in serum-free media were added to the interior of each insert. Plates were incubated for 24 hours at 37°C in 5% CO2, and media removed from the insert, which was then washed with PBS. Insert membranes were fixed with cold methanol for 10 minutes, stained with 0.5% Crystal Violet in 25% methanol for 10 mins and rinsed with water to remove excess dye. Membranes were removed from the insert, placed under a microscope and the number of cells that migrated through the porous membrane was counted.
Invasion assays were done as described above using BD BioCoat™ Matrigel™ invasion chambers (BD Biosciences, Mississauga, Canada).
5 × 103 cells/well were seeded in 96-well plates in 100 ul medium with 10 μM cisplatin and without phenol red and left to incubate for 48 hours at 37°C and 5% CO2 . After 48 hours, 10 μl MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, Oakville, Canada) was added to each well and cells were left for 4 hours. After incubation, 150 μl MTT solvent (0.1 N HCl in anhydrous isopropanol) was added to each well and mixed thoroughly by pipetting until all formazan crystals were dissolved. Colourimetric change was measured at 570 nm and background absorbance at 690 nm. Final values were obtained by subtracting OD690 nm from OD570 nm. MTT assays for siRNA optimization were done without adding cisplatin and the initial seeded cells were only incubated for 24 hours prior to MTT addition.
Pre-designed siRNA oligos for snail (cat# SASI_Hs01_00039785, duplex sequences: 5'GCCUUCAACUGCAAAUACU and 5'AGUAUUUGCAGUUGAAGGC) and slug (cat# SASI_Hs01_00159363, duplex sequences: 5'GCAUUUGCAGACAGGUCAA and 5'UUGACCUGUCUGCAAAUGC) were purchased from Sigma-Aldrich (Oakville, Canada).
Optimization of gene knockdown was done using AllStars Hs Cell Death Control siRNA (Qiagen, Toronto, Canada), a mix of siRNAs that target genes essential for cell survival. 5 × 104 cells/well were plated in 24-well plates and incubated overnight. After 24 hours cells were transfected with 7.5 ng, 19 ng, 37.5 ng or 75 ng of AllStars Hs Cell Death Control siRNA and 1.5 μl, 3 μl or 7.5 μl of HiPerFect Transfection Reagent (Qiagen, Toronto, Canada). Cells were incubated for 72 hours and cell death measured using MTT assays. Maximum cell death was achieved using 19 ng siRNA with 3 μl of transfection reagent. Optimal time points were then established using siRNA targeted against Snail and Slug. 24, 48 and 72 hours after transfection the level of snail and slug transcript was determined by qRT-PCR TaqMan assay. Optimal knockdown of both genes was seen 72 hours post transfection. Efficiency of transfection in the A2780 and A2780cis cells was determined to be 87% and 84%, respectively.
For knockdown experiments, 5 × 104 cells/well were plated in 24-well plates and incubated overnight. 19 ng of siRNA and 3 μl of HiPerFect transfection reagent were diluted in 100 μl serum-free RPMI and were incubated for 10 minutes at room temperature. Transfection complexes were then added drop-wise onto the cells. Cells were incubated for 72 hours at 37°C and 5% CO2. Media was changed as necessary. Transfection of A2780cis cells with AllStars Negative Control (Qiagen), an siRNA sequence with no homology to any known mammalian gene, was used as a control for this experiment. Cells with reduced expression were designated as follows: A2780cisSN - snail knockdown, A2780cisSL - slug knockdown, A2780cisSN/SL - both snail and slug knocked down.
Each assay was performed in triplicate. Data are expressed as the mean ± standard deviation (SD). Statistical significance of all data was evaluated using the Student's t-test, p < 0.05.
Sample preparation for proteomic analysis
Samples were lysed in RIPA buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (PIERCE, IL, USA). Cells were sonicated and lysates incubated at 4°C for 30 minutes with shaking. Supernatants were separated by centrifugation for 15 minutes at 4°C. Protein concentration was measured with a Bio-Rad DC Protein Assay kit and 30 μg of protein was used for tryptic digestion. Each aliquot was dried, dissolved in 0.5 M triethylammonium bicarbonate (TEAB), reduced by adding 2 μl of tris-(2-carboxyethyl)-phosphine (TCEP), incubated at room temperature for 1 hour, alykalted using 1 μl of methyl methane thiosulfonate (MMTS) and incubated at room temperature for 1 hour in the dark. 2 μg trypsin was added for overnight digestion at 37°C. The tryptic digest was desalted by using a C18 spin column (PIERCE, USA), dried under vaccum and resuspended in 0.1% formic acid for SRM analysis.
The LC/SRM-MS analytical system consisted of an Eksigent nanoflow HPLC (AB Sciex, USA) coupled to a 5500 QTRAP® hybrid triple quadrupole/linear ion trap mass spectrometer (AB SCIEX, USA). 1 μg of digested protein was loaded onto a trap column (0.3 mm I.D, 5 mm L), packed with 5 μm Zorbax SB-C18, 300 Å pore (Agilent, USA). Peptides were separated on a 75 μm I.D., 15 cm long nanoflow column with 15 μm spray tip (New Objectives, USA). A linear gradient profile was employed starting from 5% solvent B to 40% B in 60 minutes (solvent A was 2% acetonitrile in water with 0.1% formic acid; solvent B was 2% water in acetonitrile with 0.1% formic acid). The ion spray voltage was set at 2300 V, and source temperature at 160°C. The declustering voltage was 100 V, and collision energy value was selected for each transition as generated by MultiQuant® software (AB Sciex, USA). To ensure sensitive and reliable quantification, tryptic peptides and SRM transitions were generated by MRMPilot software (AB SCIEX, USA) based on common chemical rules of peptide fragmentation. The specificity of each peptide was verified using BLAST alignment against the NCBI-NR human protein database. Raw SRM-MS data was preprocessed using MultiQuant 2.0.2 software (AB SCIEX). A 2-point Gaussian smoothing window was applied to all transition peaks. Peak area for each transition was averaged over three replicate experiments per resistant and sensitive cell line sample; fold-change was calculated from the ratio of the average peak area in resistant cells to that in sensitive cells. A transition was discarded if peak area coefficient of variation (CV) across replicates was greater than 0.2 or peak area in any replicate was below the 25th percentile. Peptides with at least 2 transitions satisfying these constraints were conserved.