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  • Seven human thyroid cancer cell lines were evaluated, including a papillary (BHP7-13), a follicular (WRO82-1), a follicular undifferentiated (FRO81-2) and four anaplastic (8305C, 8505C, KAT18, KAT4C) cancer cell lines [21–25]. With the exception of KAT4C cells, for which no DNA short tandem repeat (STR) profile is available, all cell lines were authenticated using DNA STR profiling and stored in liquid nitrogen until use. BHP7-13, WRO82-1, FRO81-2, KAT18 and KAT4C were maintained in RPMI 1640 with sodium bicarbonate (2.0 g/L). 8305C and 8505C were maintained in MEM with sodium pyruvate (1 mmol/L) and sodium bicarbonate (2.2 g/L). 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 37°C. Dinaciclib was obtained from Selleck Chemicals and was dissolved in DMSO (Sigma) to a concentration of 10 mM and stored at -80°C until further use in vitro experiments. For the in vivo studies, dinaciclib was diluted in 20% (w/v) of 2-hydroxypropyl-β-cyclodextrin (Sigma) and stored at -30°C until use. Antibodies targeting cyclin B1, Aurora A, Mcl-1, Bcl-xL, survivin and pro-caspase-3 were purchased from Cell Signaling Technology. CDK1 and α-tubulin antibodies were obtained from Sigma. Cells were plated at 2 x 103 (BHP7-13, FRO81-2, 8505C), 1 x 104 (KAT18) or 2 x 104 (WRO82-1, 8305C, KAT4C) cells per well in 24-well plates in 1 mL media. After an overnight incubation, six serial 1:1 dilutions of dinaciclib or vehicle were added at the starting dose of 100 nM over a 4-day treatment course. Cytotoxicity was determined on day 4. Culture medium was removed and 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 (Promega) at 490 nM by spectrophotometry (Infinite M200 PRO, Tecan). Each experiment was performed in triplicate, and the results are shown as the percentage of surviving cells determined by comparing the LDH of each sample relative to control samples, which were considered 100% viable. Median-effect dose (Dm) on day 4 was calculated for each cell line using CompuSyn software [26,27]. Dm is the dose inhibiting cell viability by 50%. The effects of dinaciclib on cell cycle progression were evaluated. Cells were plated at 4 x 104 (KAT4C) or 1 x 105 cells (all other cell lines) per well in 6-well plates in 2 mL of media overnight. Dinaciclib (25 nM), a clinically relevant dose [19], or vehicle was added and incubated for 24 h, after which adherent cells were trypsinized, washed with PBS, fixed with cold 70% ethanol and incubated with RNase A (100 μg/mL; Sigma) and propidium iodide (PI, 5 μg/mL; Sigma) at 37°C for 15 min. Cell cycle distribution was assessed by DNA content detected by flow cytometry (BD FACScalibur Flow Cytometer, BD Biosciences). Each condition was performed in triplicate. The effect of dinaciclib on mitotic progression was evaluated using confocal microscopy. Thyroid cancer cells were plated at 5 x 104 (WRO82-1) or 1 x 105 (BHP7-13, 8505C) in four-well culture slides in 1 mL of media overnight. Cells were treated with dinaciclib (25 nM) or placebo for 24 h, washed with PBS, fixed in 4% paraformaldehyde (Sigma) for 15 min at room temperature, washed with PBS, permeabilized with 0.1% Triton X-100 (10 min, room temperature), washed with PBS, incubated with 4',6-diamidino-2-phenylindole (DAPI; 0.2 μg/mL, Invitrogen) for 10 min at room temperature, washed with PBS, and covered with Vectashield mounting medium (Vector Laboratories). Images were captured with Leica TCS SP8 X confocal microscopy (Leica Microsystems). Chromosomes were examined to identify mitotic cells. The expression of cyclin B1 and Aurora A was evaluated using immunofluorescence microscopy. Dinaciclib (25 nM) or placebo treated thyroid cancer cell samples were prepared as described above. Cells were then incubated with primary rabbit cyclin B1 antibody (1:200), rabbit Aurora A antibody (1:200) and mouse α-tubulin antibody (1:1000) at 4°C overnight, washed with PBS and incubated with secondary Alexa Fluor 633-conjugated goat anti-rabbit antibody (1:1000; Invitrogen) and Alexa Fluor 488-conjugated goat anti-mouse antibody (1:1000; Life Technologies) for 25 min at 37°C, washed with PBS, counterstained with DAPI, washed with PBS and covered with mounting medium. Images were acquired using Leica TCS SP8 X confocal microscopy. Cells were plated at 1 x 106 cells in 100-mm Petri dishes in 10 mL of media overnight and treated with dinaciclib at 25 nM or vehicle for the indicated periods. Cell pellets were dissolved in radio-immunoprecipitation assay buffer and protease inhibitor cocktail, vortexed and clarified by centrifugation. Total protein (40 μg) was separated by electrophoresis on 6% or 12% Tris-HCl gels, transferred to polyvinylidene 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 (PerkinElmer). Caspase-3 activity was analyzed using fluorometric assay kit (Abcam). Cells were plated at 1 x 106 cells in 100-mm Petri dishes in 10 mL of media overnight. Dinaciclib (25 nM) or vehicle was added for 24 h. Adherent cells (5 x 105) were collected, centrifuged, lysed using 50 μL of lysis buffer on ice for 10 min, incubated with DEVD-AFC substrate and reaction buffer at 37°C for 1.5 h. Caspase-3 activity was detected by spectrophotometry. The fluorescence intensity of the treated samples was compared with that of control samples to determine the fold-increase in caspase activity. Each condition was performed in duplicate. Early apoptosis was measured by Annexin V-Alexa Fluor 488 and PI staining kit (Invitrogen). Cells were plated at 1 x 105 cells per well in 6-well plates in 2 mL of media overnight and treated with dinaciclib (25 nM) or placebo for 24 h. Adherent cells were collected, washed with PBS and incubated with Annexin V-Alexa Fluor 488 and PI at room temperature in the dark for 15 min per the manufacturer’s protocol. Early apoptotic cells (Annexin V-positive, PI-negative) were detected by flow cytometry (BD FACScalibur Flow Cytometer, BD Biosciences). Each condition was performed in triplicate. Sub-G1 apoptosis was detected using flow cytometry. Cells were plated at 4 x 104 (KAT4C) or 1 x 105 cells (all other cell lines) per well in 6-well plates in 2 mL of media overnight and treated with dinaciclib (25 nM) or vehicle. Both floating cells and trypsinized adherent cells were collected at 72 h and processed as described above for cell cycle assessment. Each experiment was performed in triplicate and the proportion of apoptotic sub-G1 cells was determined by measuring the DNA content using flow cytometry. Eight-week-old athymic female nude mice from the National Laboratory Animal Center, Taiwan, were anesthetized with an intraperitoneal injection of 2% 2,2,2-Tribromoethanol (200 μl/mouse; Sigma) before implantation of thyroid cancer cells. 8505C flank tumors were established by injecting 1 x 106 cells in 100 μL of ECM gel (Sigma) into the subcutaneous flanks of nude mice. When 8505C tumors reached 5.0 mm in mean diameter, mice received daily intraperitoneal injections of vehicle (n = 13), lower-dose dinaciclib (40 mg/kg, n = 13) or higher-dose dinaciclib (50 mg/kg, n = 7). These doses were chosen based on a previous report [15]. Tumor dimensions were serially measured with electronic calipers, and the volumes were calculated by the following formula: a x b2 x 0.4, where a represents the largest diameter and b is the perpendicular diameter. The body weight of each animal was followed as a marker of toxicity. Tumor levels of CDK1, cyclin B1, Aurora A, Mcl-1, Bcl-xL, survivin and pro-caspase-3 were evaluated in mice treated with daily intraperitoneal injections of lower-dose dinaciclib (40 mg/kg) by Western blot analysis. At indicated periods, animals were euthanized with carbon dioxide, and the tumors were harvested, mixed with protein extraction buffer (GE Healthcare), homogenized and sonicated on ice. After centrifugation, clarified supernatants were aliquoted and stored at -80°C for Western blotting. This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Chang Gung Memorial Hospital, and the protocol was approved by the Committee of Laboratory Animal Center at the Chang Gung Memorial Hospital, Linkou (permission No: 2013121401). Housing and care of mice were provided by the Laboratory Animal Center, Chang Gung Memorial Hospital, Linkou. Animals were given ad libitum access to food and water. The physical condition of mice was monitored by our animal care personnel on a daily basis. In addition, the investigators checked the conditions of the mice at least once a week before treatment, and daily during the treatment period. The humane endpoints were a tumor diameter ≥ 2.0 cm, significant weight loss (20% of pre-experiment body weight), weight loss to a final weight of 16 g, very slow breathing rate, shallow or labored breathing pattern, decreased activity, poor response to handling, absence of grooming, social isolation, hunched posture, shivering, and muscle atrophy. The method of euthanasia was CO2 exposure for 10 min, at a 20% fill rate of cage volume/min. Comparisons were performed when appropriate using two-sided Student’s t tests (Excel, Microsoft). P < 0.05 was considered statistically significant. Results were expressed as mean ± SE.
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