Undifferentiated human anaplastic thyroid carcinoma SW1736 cells were provided by Professor Michael Derwahl (Institute of Endocrinology, Hedwig Hospital, Humboldt University, Berlin, Germany); this cell line has a tolerance to isotope treatment (16). The cells were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) in an incubator at 37°C with 5% CO₂.

ATRA (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was dissolved in ethanol to 10 µmol/l as stock solution and stored in the dark. SW1736 cells were seeded at a density of 5×10⁴ cells/well for 48 h, followed by ATRA treatment (1 µmol/l) for 5 days. The negative control cells were treated with 1% ethanol. All cells were kept at 37°C in the dark using aluminum foil. SW1736 cells stably transfected with β-catenin shRNA were used as positive controls.

The recombinant plasmid pSUPER-β-catenin-shRNA was previously constructed (17). It was transfected into SW1736 cells using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). SW1736 cells transfected with pSUPER-β-catenin-shRNA were screened by 400 µg/ml puromycin for 4 weeks to select positive clones. Finally, cells were screened and cultured in medium with 200 µg/ml of puromycin to establish the stable transfected cell line.

SW1736 cells treated with 1 µmol/l of ATRA or 1% ethanol for 5 days were seeded onto a 96-well culture plate. Each well was loaded with 180 µl of cell suspension containing 1×10⁴ cells. After 24, 48 and 72 h, the supernatant was discarded and cells were treated with 150 µl DMEM and 50 µl MTT solution (2.5 mg/ml; Sigma-Aldrich; Merck KGaA). The MTT solution was discarded following 4 h of treatment, and the cells were mixed with 150 µl dimethyl sulfoxide at low-speed vortex for 10 min. The absorbance of each well was measured at 570 nm using DNM-9602 ELISA spectrometer (Perlong Medical Equipment Co., Ltd., Beijing, China).

Transwell chambers (Merck KGaA) coated with Matrigel (Sigma-Aldrich; Merck KGaA) were placed into 6-well plates and air-dried under a sterile laminar flux hood. DMEM medium (700 µl) containing 10% FBS was added to the lower chamber. Cell suspension (300 µl) containing 5.0×10⁴ SW1736 cells treated with 1 µmol/l ATRA or 1% ethanol, but without FBS, for 5 days was added to the upper chamber. After 24, 48 and 72 h, the Transwell chambers were washed with phosphate-buffered saline (PBS). The upper microporous membrane was cleaned with cotton swab. Invading cells attached to the lower microporous membrane were fixed with 2% paraformaldehyde for 30 min and stained with hematoxylin. Cell counting was performed by two independent investigators blind to grouping.

The in vitro iodine uptake assay was performed as previously described (18). Briefly, SW1736 cells treated with 1 µmol/l ATRA or 1% ethanol for 5 days were seeded onto 6-well plates (5.0×10⁴ cells/well). After the cells had attached to the bottom surface, the supernatant was discarded. Cells were washed with Hank's balanced salt solution (HBSS) and incubated with 3.7 kBq of ¹²⁵I (Thermo Fisher Scientific, Inc.) for 20 min followed by three washes with ice-cold HBSS. Cells were then incubated with 1 ml of ethanol for 20 min. The ethanol solution was collected into test tubes and the radioactivity per minute was measured using a WIZARD γ counter (Perkin-Elmer Life Sciences, Waltham, MA, USA).

SW1736 cells treated with 1 µmol/l ATRA or 1% ethanol for 5 days were lysed with 1% NP-40 lysis buffer (Sigma-Aldrich; Merck KGaA) at 4°C for 30 min. Total proteins were collected after centrifugation at 25,000 × g for 10 min at 4°C. Proteins were quantified using Coomassie brilliant blue G-250. Samples of 20–50 µg per well were separated on 8–12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk for 2 h followed by incubation with primary antibodies against β-catenin (dilution: 1:4,000; cat. no. ab16051; Abcam, Cambridge, MA, USA), phosphorylated (p-)β-catenin Ser45 (dilution, 1:1,000; cat. no. PA5-17685; Invitrogen; thermo Fisher Scientific, Inc.), p-β-catenin Y654 (dilution, 1:1,000; cat. no. ab59430, Abcam), glycogen synthase kinase-3β (GSK-3β) (dilution, 1:10,000; cat. no. ab75814, Abcam), p-GSK-3β Ser9 (dilution, 1:10,000; cat. no. ab75814; Abcam), E-cadherin (dilution, 1:1,000; cat. no. sc-7870; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), cytokeratin 18 (CK18) (dilution: 1:1,000; cat. no. sc-28264; Santa Cruz Biotechnology, Inc.), vimentin (dilution: 1:1,000; cat. no. sc-5565; Santa Cruz Biotechnology, Inc.), urokinase-type plasminogen activator (uPA) (dilution: 1:1,000; cat. no. sc-14019; Santa Cruz Biotechnology, Inc.) and its receptor uPAR (dilution: 1:1,000; cat. no. sc-10815; Santa Cruz Biotechnology, Inc.), and fibronectin (dilution, 1:1,000; cat. no. sc-9068; Santa Cruz Biotechnology, Inc.) at 4°C overnight. The next day, the membranes were extensively washed with Tris-buffered saline and incubated with goat anti-rabbit secondary antibody (dilution: 1:30,000; cat. no. A9919; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 2 h at 4°C. The bands were visualized by electrochemiluminescence (Pierce; Thermo Fisher Scientific, Inc.). Densitometry images were analyzed using ImageJ software (Rawak Software, Inc., the Tomancak Laboratory, Dresden, Germany).

SW1736 cells (2×10⁶) were treated with 1 µmol/l ATRA or 1% ethanol for 5 days, and SW1736 cells stably transfected with β-catenin shRNA were suspended in 200 µl of culture medium and implanted subcutaneously into the left forelimb of 4–6-week-old male SCID mice (22–25 g body weight). Intervention was applied after the subcutaneous tumor size reached 50 mm³. The experimental mice (alcohol, β-catenin shRNA and ATRA groups) were intraperitoneal injected with 37 MBq of ¹³¹I (Thermo Fisher Scientific, Inc.) once on the first day of treatment. The control mice (ATRA and alcohol groups) were intraperitoneally injected with 0.1 ml of normal saline (NS) instead (ATRA+NS and Alcohol+NS). There were 10 mice in each group (supplied by Beijing Huafukang Biotechnology Co., Ltd., Beijing, China). The ¹³¹I intervention was commenced on the first day of the experiment. Subcutaneous tumor size was measured every 5 days using a caliper (volume=long diameter × short diameter² + 2). Mice were sacrificed by CO₂ inhalation after 35 days of observation. Tumors were collected and weighed. All procedures and animal experiments were approved by the Animal Care and Use Committee of Beijing Jishuitan Hospital (Beijing, China).

All statistical analyses were conducted using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation (SD) from three independent experiments performed in triplicate. Statistical significance was evaluated by one-way analysis of variance, with the least significant difference test for post-hoc analysis. P<0.05 was considered to indicate a statistically significant difference.

After 35 days of ¹³¹I treatment, ATRA-pretreated tumor volume and weight were decreased compared with the alcohol+¹³¹I group (163.32±19.57 vs. 332.06±21.37 mm³; 0.35±0.14 vs. 0.67±0.23 g; both P<0.05) (Fig. 1). Similar results were observed in the β-catenin-shRNA+¹³¹I group (116.3±14.4 mm³ and 0.26±0.11 g, both P<0.05 vs. the alcohol+¹³¹I group). There were no differences in tumor volume and weight among the tumors treated with ATRA+NS, Alcohol+NS, and Alcohol+¹³¹I groups (all P>0.05). Furthermore, there were no differences in the tumor volume and weight between the tumors treated with ATRA+¹³¹I and β-catenin-shRNA+¹³¹I (both P>0.05) (Fig. 1).

The iodine uptake assay revealed that ATRA increased the uptake of iodine by SW1736 cells compared with the alcohol control group (1.4±0.2 vs. 0.4±0.2×10³/min; P<0.01) (Fig. 2A). Similar results were observed in the β-catenin-shRNA cells compared with the no transfection group (1.7±0.3 vs. 0.6±0.2×10³/min; P<0.01).

MTT and Transwell invasion assays indicated that ATRA treatment decreased the cell proliferation (Fig. 2B) and invasion (Fig. 2C) compared with untreated and alcohol control cells (P<0.05). β-catenin-shRNA cells showed results that were similar to the ATRA-treated cells.

Western blot analysis revealed that ATRA treatment decreased the expression of t-β-catenin, p-β-catenin Ser45, p-β-catenin Y654 and p-GSK-3β, and increased the expression of t-GSK-3β compared with untreated and alcohol control cells (Fig. 3A). Similar results were observed in β-catenin-shRNA cells.

Western blot analysis also revealed that ATRA treatment increased the expression of NIS, E-cadherin and CK18, and decreased the expression of vimentin, uPA, uPAR and fibronectin compared with untreated and alcohol control cells (Fig. 3B). Similar results were observed in β-catenin-shRNA cells.