Thyroid Cancer and NVP-BEZ235 paclitaxel showed synergistic effects in all cell lines. The combination of BEZ235 and irinotecan had enhanced cytotoxic effects at higher doses, but revealed limited or no beneficial therapeutic effects at lower doses. The combination of BEZ235 plus etoposide displayed little additional cytotoxicity. C, the CI of BEZ235 and each chemotherapeutic agent was calculated using CompuSyn software. BEZ235 plus paclitaxel exhibited synergistic effects in all cell lines, with CI,0.8 at most conditions. BEZ235 plus irinotecan had synergistic to antagonistic effects in 8305C, 8505C and KAT18, and antagonistic effects in KAT4C. BEZ235 plus etoposide demonstrated mostly antagonistic effects in all cell lines. The horizontal dash lines at CI = 1 were drawn for discrimination of synergism (CI,1) and antagonism (CI.1). D, the typical ranges of DRI values for chemotherapeutics in combination with BEZ235. With the presence of BEZ235, paclitaxel mostly had the greatest DRIs in 8505C, KAT4C and KAT18, and also demonstrated favorable DRIs in 8305C. doi:10.1371/journal.pone.0046726.g006 sensitivity of BEZ235, because both sensitive (8305C) and less sensitive (WRO82-1) cell lines harbor the mutations. Exploration of genetic changes of mTOR, the major target of BEZ235, might someday provide information that may predict therapeutic efficacy. Inhibition of mTORC1 may activate the MAPK pathway through a PI3K-dependent feedback loop [45], which explains why BEZ235 was seen here to activate ERK1/2 in thyroid cancer cells. Combining BEZ235 with an inhibitor targeting MAPK pathway may be a potential approach to enhance therapeutic efficacy. Recently, the combination of a pan-RAF inhibitor and BEZ235 was shown to induce cell cycle arrest at G0/G1 phase, and had beneficial combination effects in treating thyroid cancer [46]. Similarly, in combination of mTOR and MEK inhibitors also demonstrated therapeutic advantage in treating thyroid cancer, providing further evidence that targeting both PI3K/ mTOR and MAPK pathways is a potential therapeutic strategy for this disease [47,48]. Materials and Methods Cell lines Eight cell lines were obtained, including a papillary (BHP7-13), a follicular (WRO82-1), a follicular undifferentiated (FRO81-2), four anaplastic (8505C, 8305C, KAT4C, KAT18) and a medullary (TT) human thyroid cancer [23–25]. All cell lines except KAT4C were authenticated using DNA (short tandem repeats) profiling and stored in liquid nitrogen [26]. BHP7-13, WRO82-1, FR081-2, KAT4C and KAT18 were maintained in RPMI 1640 with sodium bicarbonate (2.0 g/L). 8505C and 8305C were maintained in MEM with sodium pyruvate (1 mmol/ L) and sodium bicarbonate (2.2 g/L). TT was maintained in F12K. All media contained 10% FCS, 100,000 units/L penicillin and 100 mg/L streptomycin. All cells were maintained in a 5% CO2 humidified incubator at 37uC. Pharmacologic agents BEZ235 is a dual PI3K/mTOR inhibitor described previously [17], and was a generous gift from Novartis Pharma AG (Basel, Switzerland). BEZ235 (10 mmol/L) was dissolved in DMSO (Sigma) and stored at 230uC until in vitro experiments. For in vivo studies, BEZ235 was diluted with 1-Methyl-2-pyrrolidone (NMP; Sigma) and poly(ethylene glycol) (average Mn 285–315; Sigma) (1:9 v/v) to a final concentration of 21.5 mg/ml. Paclitaxel, irinotecan, etoposide, 5-Fluorouracil (5-FU) and doxorubicin were obtained from Sigma Chemical Co. Paclitaxel (320 mmol/L), irinotecan (16 mmol/L), etoposide (5 mmol/L) and 5-FU (40 mmol/L) were dissolved in DMSO and doxorubicin (5 mmol/L) was dissolved in PBS and stored at 230uC until use. Antibodies Antibodies targeting p-AKT (Thr308), p-AKT (Ser473), AKT, p-4E-BP1 (Thr70), p-4E-BP1 (Thr37/46), 4E-BP1, p-S6 ribosom- al proteins (Ser235/236), p-S6 ribosomal protein (Ser240/244), S6 ribosomal protein, p-ERK1/2 (Thr202/Tyr204), ERK1/2, p53, p21, p27, cyclin D1, caspase-3 and proliferating cell nuclear antigen (PCNA) were from Cell Signaling Technology. a-tubulin antibody was from Sigma. Cytotoxicity assays Cells were plated at 26104 cells per well in 24-well plates in 1 mL media. After overnight incubation, the agents or vehicle were added at the indicated concentration. Six serial 1:1 dilutions were tested starting at the following doses: BEZ235 at 100 nmol/ L, paclitaxel at 68 nmol/L (8305C, 8505C and KAT4C) and 20 nmol/L (KAT18), irinotecan at 8 mmol/L, etoposide at 10 mmol/L, 5-FU at 20 mmol/L and doxorubicin at 1 mmol/L over a 4-day treatment course. Cytotoxicity was determined daily for BEZ235 group and on day 3 and 4 for the chemotherapy groups. Cells were washed with PBS and lysed with Triton X-100 (1.35%, Sigma) to release intracellular lactate dehydrogenase (LDH), which was quantified with a Cytotox 96 kit (Progmega) at 490 nM by spectrophotometry (BT-MQX200R, Bio-Tek Instru- ments). Each experiment was performed in triplicate and results are shown as the percentage of surviving cells determined by comparing the LDH of each sample relative to control samples which are considered 100% viable. Median effect doses (Dm) on day 4 were calculated for each cell line and drug using CompuSyn software [27]. For combination therapy experiments, ATC cells were treated with BEZ235 and a chemotherapeutic drug (paclitaxel, irinotecan or etoposide) at a fixed dose ratio. Cells were incubated with vehicle, BEZ235, chemotherapeutic agent, or both simultaneously for a 4-day course and cytotoxicity was measured. Five serial 1:1 dilutions were examined at the following starting doses for 8305C, 8505C, KAT4C and KAT18: BEZ235 at 26.8, 37.2, 15.6 and 26.4 nmol/L, paclitaxel at 52.8, 22.4, 28 and 17.6 nmol/L, irinotecan at 13.6, 14, 13.6 and 11.6 mmol/L, etoposide at 11.2, 2.56, 6.8 and 5.2 mmol/L, respectively. The doses chosen were based on the Dm determined previously. Western blots Cells were plated at 86105 cells in 100-mm Petri dishes in 8 mL media overnight and treated with BEZ235 or vehicle for indicated periods. Cells were plated overnight for baseline expression of untreated cells. Cell pellets were dissolved in radio-immunopre- cipitation assay buffer and protease inhibitor cocktail, vortexed and clarified by centrifugation. Total protein (20–50 mg) was electrophoresed on 10–12% Tris-HCl gels, transferred to poly- vinylidene difluoride membranes, blocked, and exposed to primary antibodies followed by a secondary antibody conjugated to horseradish peroxidase. Signals were developed using an enhanced chemiluminescence kit (Bionovas Biotechnology). Band density was imaged and quantified using Molecular Imager VersaDoc MP 4000 system (Bio-Rad). The ratios of p- AKT, p-4E-BP1, p-S6 ribosomal protein and p-ERK1/2 to the correlated total protein, and the ratios of p53, p21, p27 and cyclin D1 to a-tubulin in each cell line were calculated. Relative PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e46726