Differential Expression of mTOR Signalling Components in Drug Resistance in Ovarian Cancer

Materials and Methods

Cell culture. All cell culture reagents were obtained from Invitrogen (Paisley, UK) unless stated otherwise. PEO1 and SKOV-3 paclitaxel-sensitive and -resistant ovarian cancer cell lines, respectively, were developed by Dr Helen Coley, University of Surrey (10). For PEO1, RPMI-1640 10% foetal calf serum (FCS), 5% penicillin-streptomycin and 2 mM glutamine were used for cell culture at 37°C with 5% CO₂. The SKOV-3 cell line was cultured in minimum essential medium/Earl's salts non-essential amino acid medium supplemented with 10% FCS, L-glutamine and the penicillin-streptomycin mixture at 37°C with 5% CO₂.

Chemosensitivity testing for the assessment of sensitivity to the mTOR inhibitor rapamycin. Rapamycin was obtained from Sigma Aldrich (Poole, UK) and made up in sterile water as a stock solution. XR9576 (Tariquidar) was obtained from Xenova PLC (Slough, UK) and made up as a stock solution in dimethyl sulfoxide (DMSO) and stored as frozen aliquots. PEO1 cell lines were set up in 96-well plates at a cell density of 3×10⁴ per ml and allowed to attach for 24 h. Rapamycin diluted in tissue culture medium was then added in increasing concentration in quadruplicate. Cells were then placed back in the incubator for 72 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml in phosphate buffered saline; PBS) was added to each well in 20 μl and left for 3-4 h to allow formazan crystal formation. Wells were aspirated and 200 μl of DMSO added to dissolve formazan crystals. The resulting purple coloration was measured at 540 nm in a plate reading spectrophotomer. Dose–response curves were constructed and used to determine the IC₅₀ dose (defined as the drug dose that gives rise to 50% loss of cell viability compared with untreated control wells). In limited experiments, we used the MDR modulator XR9576 at 100 nM in combination with rapamycin in order to ascertain any MDR reversal effects.

RNA isolation, cDNA synthesis and PCR. Total ribonucleic acid was isolated using an RNA extraction kit (Sigma Aldrich), according to the manufacturer's instructions. RNA concentration was determined by spectrophotometric analysis (NanoDrop; Thermo Scientific, UK) and agarose gel electrophoresis. RNA (500 ng) was reverse-transcribed into cDNA using 5 IU/μl RNase H reverse transcriptase (Invitrogen). PCR amplification was performed using Taq polymerase (Invitrogen) and oligonucleotide primers as previously described (24). A total of 28 cycles for each gene were performed, consisting of a denaturing step at 94°C for 30 s, extension at 60°C for 1 min and elongation at 72°C for 1 min.

Quantitative RT-PCR. Relative expression of the genes of interest was assessed by quantitative PCR (Q-PCR) on an ABI Prism 7900HT Sequence detection system (Applied Biosystems) using SYBR® Green-PCR reaction mixture (Sigma Aldrich) and the primers as follows: DEPTOR (202 bp): forward: 5′-caccatgtg tgtgatgagca-3′, reverse: 5′-tgaaggtgcgctcatacttg-3′; RICTOR (117 bp): forward: 5′-ggaagcctgttgatggtgat-3′, reverse: 5′-ggcagcctgtttta tggtgt-3′; RAPTOR (170 bp): forward: 5′-actgatggagtccgaaatgc-3′ reverse: 5′-tcatccgatccttcatcctc-3′; 18S RNA (155 bp): forward: 5′-aaacggctaccacatccaag-3′, reverse: 5′-cctccaatggatcctcgtta-3′.

As a negative control, distilled water was used in place of the cDNA. RNAs were assayed from two to three independent biological replicates. The RNA levels were expressed as relative RQ values, using the parental cell line as calibrator. The Delta Delta Ct method was employed for comparing relative expression results between treatments in Q-PCR (25).

Immunofluorescent analysis. SKOV-3 and PEO1 cells were fixed in 4% paraformaldehyde for 10 min prior to washes in PBS and incubation with 10% bovine serum albumin (BSA) for 1 h. Cells were incubated for 1 h with an mTOR antibody (Santa Cruz Biotechnology, USA) at a 1:100 dilution in 1% BSA PBS. Cells were washed with PBS prior to an incubation with anti-rabbit IgG-fluorescein isothiocyanate (FITC)-conjugated antibody (Santa Cruz Biotechnology, USA) for 1 h. Slides were washed with PBS and mounted in Vectashield® Mounting Medium (Vector Labs) containing the dye 4,6-diamido-2-phenylindole (DAPI) to counterstain nuclei. Images were captured using a Plan Apo Neofluor X63 NA 1.25 oil objective (Zeiss) on a Zeiss Axiovert 200M microscope and viewed using AxioVision software, at set exposure times.

Protein extraction from cultured cells. SKOV-3 and PEO1 cells were cultured in 6-well dishes until 80% confluency. Cells were lysed with 300 μl 2× Laemmli Buffer (Sigma Aldrich) and denatured for 5 min at 100°C before being cooled on ice.

Western immunoblotting. Samples were separated on an SDS-10% polyacrylamide gel and the proteins were transferred to a nitrocellulose membrane. The membrane was blocked in TBS containing 0.1% Tween-20 and 5% dried milk powder (wt/vol), for 1 h at room temperature. After a brief wash with PBS-0.1% Tween-20, the nitrocellulose membranes were incubated with primary antibodies against phospho-mTOR (Ser2448), total mTOR, phospho-p70S6K (serine371/threonine389), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signalling, USA; Sigma Aldrich). The primary antisera were used at a 1:1000 dilution in PBS-0.1% Tween-20 overnight at 4°C. The membranes were washed for 30 min with PBS-0.1% Tween-20, before incubation with the secondary anti-rabbit horseradish peroxidase-conjugated immunoglobulin (1:2000) for 1 h at room temperature and further washing for 30 min with PBS-0.1% Tween-20. Antibody complexes were visualised as previously described (26).

Statistical analysis. For the quantitative PCR, the following equations were used: ΔCt=Ct_{(gene of interest)}-Ct_{(house keeping gene)}, ΔΔCt=ΔCt_(sample)– ΔCt_(calibrator), relative quantity (RQ)=2_–ΔΔCt. RQ value was set at 1 for the parental SKOV-3 and PEO1 cells when compared to paclitaxel-resistant (TaxR) ones. Data are reported as the mean±SD of three or four measurements. Statistical analysis was performed by Student's t-test and by ANOVA. P<0.05 was regarded as significant.