1 2 3 4 Materials and Methods Cell culture BHP 2-7 and Nthy-ori 3-1 cells were grown in RPMI1640 medium (Hyclone) 5 supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine, and 100 U/mL 6 penicillin-streptomycin. K1 cell was grown in DMEM:Ham s F12:MCDB 105 (2:1:1) 7 supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine, and 100 U/mL 8 9 10 penicillin-streptomycin. All cells were maintained at 37 C, in an atmosphere of 5% CO 2. Oncogene analysis and cell proliferation assay Total RNAs were prepared from cells cultured using RNeasy kit (Qiagen). RNA (2 µg) 11 was reverse-transcribed into 40 µl cdna using QuantiTect Reverse Transcription Kit 12 (Qiagen). Primer design was performed using Primer Express 2, and the sequences are 13 presented in supplemental table 1. Reactions were performed in 50 µl of a PCR solution 14 containing 4 µl cdna, 1 µm primer with buffers from Hotstar Taq TM plus DNA polymerase 15 kit (Qiagen). PCR products were visualized by electrophoresis in 2% agarose gel. Products 16 were submitted to direct sequencing. Mutations of RET/PTC1, BRAF, and NRAS (sequence 17 was seen in supplemental table 1) were confirmed by sequencing of antisense strands. 18 Cell counting kit (CCK8) was used to detect cell proliferation. Briefly, cells were grown to 19 a confluency of 90% in 25 cm 2 cell culture flasks and passed into 96-well plates (4 10 3 20 cells/well). Each well contained 100 µl of the culture medium supplemented with various 21 concentrations of sorafenib (5nM, 50nM, 500nM, 5µM) and cabozantinib (5nM, 50nM, 22 500nM, 5µM for BHP 2-7 cell,500nm, 2.5µM,5 µm,10 µm,25 µm for K1 cell) or a 23 concentration of DMSO equivalent to the highest amount of stock sorafenib or cabozantinib 24 solution added to serve as a control. Every 24 h, the medium was removed and 100 µl of
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 fresh RPMI1640 medium containing the appropriate concentration of sorafenib, cabozantinib or DMSO was added. After incubation with sorafenib, cabozantinib or DMSO for either 24, 48, or 72 h, 10 µl of the CCK8 reagent was added to each well, and the plate was placed in the 5% CO 2 incubator at 37 C for an additional 2 4 h. The optical density (OD) was then read at 490 nm using a microplate reader (Bio-Rad). The experiments were performed in triplicate, with the percentage cell survival calculated as follows: At each testing concentration, cell growth was calculated as "100 (T - T 0 )/(C - T 0 )." The absorbance of the wells treated with sorafenib or cabozantinib after a 48-h period of exposure was T, the absorbance with RPMI1640 medium only was T 0, and the control absorbance with BHP 2-7 cell dissolution in DMSO only was C. IC 50 was determined using Prism 3.0 (GraphPad Software). Cell cycle analysis and Annexin V-FITC apoptosis assay 1.0 10 6 cells were grown in 25 cm 2 cell culture flasks overnight and incubated with sorafenib (0.05 and 0.5 μm for BHP 2-7 cells, 2.5 and 5 μm for K1 cells), cabozantinib (0.05 and 0.5 μm for BHP 2-7 cells, 5 and 10 μm for K1 cells) or DMSO for 24 h, then washed twice with cold phosphate-buffered saline (PBS), and transferred by trypsinization to a 15 ml tube containing 300 µl cold PBS and 700 µl cold dehydrated alcohol. After cells were washed, DNA was stained with 50 mg/ml propidium iodide (PI) solution (0.02 mg/ml RNase A, 1.0 mg/ml sodium citrate dihydrate, 0.1 mg/ml PI, and 0.3% Triton X-100) at room temperature for 30 min protected from light. Fluorescence-activated cell sorting (FACS) was performed using flow cytometry (Becton Dickinson, USA). Cell cycle analysis was performed by ModFit LT software (Verity Software House, Inc.).
47 48 49 50 51 52 53 54 55 56 In apoptosis assay, cells (2 10 5 ) were grown in 6-well plates overnight and incubated as discribed in cell cycle analysis, then washed twice with cold PBS, and transferred by trypsinization to a 15 ml tube containing 1 ml culture medium and 1 ml cold PBS. The samples were analyzed by flow cytometry (Becton Dickinson, USA). Real-time quantitative RT-PCR analysis and Western blot analysis The RNeasy kit (Qiagen) was used according to the manufacturer s instructions to extract total RNA from cells cultured with or without sorafenib or cabozantinib. Two microgram of RNA was then reverse transcribed in a 40 μl reaction volume by QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer s instructions. The complementary DNAs (cdnas) were then diluted 1:20 in deionized water and amplified on an ABI 57 58 Prism-7500 system by using Quantifast R SYBR R Green PCR kit (Qiagen). In accordance with the manufacturer s instructions, each tube (reaction mixture volume: 20 µl) contained 59 60 61 62 63 64 65 66 67 68 69 0.4 µl of cdna, 10.0 µl Quantifast R SYBR R Green PCR master mix, and 1.0 µl of forward primer and reverse for each of the genes evaluated. Gene of 18S was served as endogenous control. The PCR program consisted of an initial step at 95 C for 5 min followed by a 40-cycle melting step at 95 C for 10 s and an annealing elongation step at 60 C for 30 s. All amplification reactions were performed in triplicate. Results were determined by the 2 - ΔΔCt method and expressed as relative expression normalized to a sample of control cells. Primer design was performed using Primer Express 2 as follows: NIS, forward 5 -gta gaa gac ctc atc aaa cct cgg-3 and reverse 5 -gga gcc ctg aag gac acc tc-3 ; Tg, forward 5 -gtg atc gat ggc cac ttc ct-3 and reverse 5 -ccg gcc tcg act ttc ctc-3 ; TPO, forward 5 -ctg tct gtc acg ctg gtt atg g-3 and reverse 5 -ggc gta cat ggc ggt gtc-3 ; TSHR, forward 5 -cta tag atg tga ctc tgc agc agc t-3 and reverse 5 -aaa tgc caa gga act tta gga gg-3 ; GLUT1, forward 5 -gga cct caa att
70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 tca ttg tgg g-3 and reverse 5 -cat cga agg tcc ggc ctt-3 ; GLUT3, forward 5 -tgt tcc acg ctc atg act gtt t-3 and reverse 5 -gct gaa gag ttc ggc cac aa-3.real-time quantitative RT-PCR analysis was performed to evaluate the expression of thyroid genes on an ABI Prism 7900HT Sequence Detector (Applied Biosystems, Foster City, CA), using SYBR GreenER qpcr SuperMix according to the instructions of the manufacturer (Applied Biosystems). For Western blot analysis, cellular proteins collected from BHP 2-7 cells or K1 cells treated with various concentrations of sorafenib or cabozantinib for 2 h were electrophoresed on a 6% or 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a PVDF membrane (Millipore). The membrane was incubated in nonfat milk/bsa for at least 1 h. Then Following this, the membrane was incubated with rabbit/mouse monoclonal antibodies to p-ret, p-erk1/2, p-akt, NIS, Tg, TPO, TSHR, GLUT1, and GLUT3 overnight. The membrane was washed twice in Tris-Buffered Saline Tween-20 (TBST) and then incubated with anti-rabbit IgG, HRP-linked antibody/anti-mouse IgG, and HRP-linked antibody for 1 h, and subsequently washed for 1 h in TBST. The proteins were illuminated with Potent ECL kit. Membranes were stripped (Beyotime stripping buffer) and reblotted with rabbit RET, ERK 1/2 and AKT and treated the same as above. RET and p-ret (Santa Cruz Biotechnology, Inc, USA). Radioiodine and FDG uptake assay In radioiodine uptake assay, BHP 2-7 cells or K1 cells and normal thyroid cells (Nthy-ori 3-1) were seeded in 6-well plates (5 10 4 cells per well) and BHP 2-7 cells were treated with the sorafenib (2.5 μm) or cabozantinib (0.5 μm), and K1 cells were treated with sorafenib (5 μm) or cabozantinib (10 μm) for 96 h. After 96 h, one well was counted for cell number for
92 each group, and the remaining wells were cultured with 2 ml RPMI 1640 medium containing 93 74 kbq Na 125 I for 1 h at 37 with 5% CO 2. To determine NIS-specific uptake, a group of 94 wells were preincubated with 300 μm NaClO 4 (Sigma) for 30 min before the start of 95 incubation with Na 125 I. The medium was subsequently aspirated, and cells were washed twice 96 with ice-cold PBS. Then cells were lysed with 0.3 mol/l sodium hydroxide on ice. Thereafter, 97 the radioactivity in cell lysates was measured using an automated NaI(Tl) well counter. 98 Experiments were performed in triplicate and repeated at least twice. 99 In FDG uptake assay, BHP 2-7 cells treated with sorafenib (2.5 μm) or cabozantinib (0.5 μm), 100 K1 cells treated with sorafenib (5 μm) or cabozantinib (10 μm) for 96 h were seeded in 101 6-well plates (1 10 5 cells per well). After 96 h, thirty minutes before adding 18 F-FDG, media 102 were changed to glucose-free RPMI1640 containing 10% FBS. One well was counted for cell 103 number for each group, and the remaining wells were incubated for 1 h at 37 C in each of the 104 following conditions: 20 μci 18 F-FDG alone, 20 μci 18 F-FDG + 5mmol/L glucose + 50 105 μmol/l cytochalasin B. The cells were rinsed in cold PBS, harvested in 500 μl of 1%SDS + 106 107 108 109 110 0.3 mol/l sodium hydroxide and counted by a gamma counter. Supplemental Table 1: Sequences of NRAS, BRAF, and RET/PET1 Primer NRAS BRAF RET/PET1 Sequence forward 5 -agc ttg agg ttc ttg ctg g-3 reverse 5 -atc cgc aaa tga ctt gct att a-3 forward 5 -atc cac aga gac ctc aag agt aat a-3 reverse 5 -tcc tcg tcc cac cat aaa a-3 forward 5 -aga tag agc tgg aga cct aca aac-3 reverse 5 -ggt ggt tga cct gct tca g-3 Supplemental figure 1. Sequence chromatogram of PCR product from BHP 2-7 or K1 cells. 111 (A) BHP 2-7 cells displaying RET/PTC1 rearrangement banding pattern present acc atc gag
112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 gat with wild type gene of BRAF and NRAS. (B) K1 cells displaying BRAF V600E mutant banding pattern present tac aga gaa a with wild type gene of NRAS. Supplemental figure 2. Growth inhibition curves for sorafenib-treated or cabozantinib-treated BHP 2-7 or K1 cells. BHP2-7 cells were treated with sorafenib (A) or cabozantinib (B) at concentrations of 0.005, 0.05, 0.5 and 5 μm for 1, 2, and 3 days. K1 Cells were treated with sorafenib (C) (0.005, 0.05, 0.5 and 5 μm) or cabozantinib (D) (0.5, 2.5, 5, 10 and 25 μm) for 1, 2, and 3 days. Dimethylsulfoxide (DMSO) was used as the control. Cell proliferation was plotted relative to the control. Supplemental figure 3. Cell cycle analysis of K1 cells treated with sorafenib or cabozantinib. K1 cells were treated with 2.5 and 5 μm sorafenib (A) or 5 and 10 μm cabozantinib for 24 h (B), respectively. Cells treated with dimethyl sulfoxide (DMSO) were used as a control. After flow cytometry, the percentage of cells in the G1/G0, G2-M, and S phases were shown. Supplemental figure 4. Apoptosis analysis of K1 cells treated with sorafenib or cabozantinib by Annexin V-FITC detection kit. K1 cells were treated with 2.5 and 5 μm sorafenib or 5 and 10 μm cabozantinib for 24 h, respectively. Cells treated with dimethyl sulfoxide (DMSO) were used as a control. After flow cytometry, the percentage of apoptosis cells (A) and dead cells (B) were shown. *, P < 0.05. Supplemental figure 5. Western blot demonstrating the dose-dependent effects of sorafenib and cabozantinib on the MEK/ERK pathway in K1 cells. Panels were assayed with different antibodies as indicated along the right side of the panels. (A) Lanes 1 5 are cytoplasmic extracts from K1 cells treated with dimethyl sulfoxide (DMSO), 0.5 μm, 2.5
134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 μm,5 μm and 10 μm of sorafenib. Sorafenib blocked the phosphorylation of ERK1/2 at the concentration of 10 μm. (B) Lanes 1-4 are cytoplasmic extracts from K1 cells treated with DMSO, 5μM, 10 μm and 20 μm of cabozantinib. Supplemental figure 6. Effects of sorafenib and cabozantinib on the mrna levels of sodium iodide symporter (NIS), thyroglobulin (Tg), and glucose transporter isoforms (GLUT1 and GLUT3) genes in K1 cells. Data are presented as means ± SD. *, P <0.05 for comparison with control. Supplemental figure 7. Western blot demonstrating the dose-dependent effects of sorafenib and cabozantinib on the protein levels of sodium iodide symporter (NIS), thyroglobulin (Tg), thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO), glucose transporter isoforms (GLUT1 and GLUT3) in K1 cells. Panels from top to bottom were evaluated with different antibodies as indicated along the right side of the panels. GAPDH was used as positive control. (A) Lanes 1-3 are cellular extracts from K1 cells treated with DMSO, 2.5 μm and 5 μm of sorafenib. (B) Lanes 1-3 are cellular extracts from K1 cells treated with DMSO, 5 μm, and 10 μm of cabozantinib. Supplemental figure 8. Radioiodine and 18 F-FDG uptake assay in K1 cells treated with sorafenib or cabozantinib. (A) 125 I uptake in K1 cells induced by 5 μm of sorafenib or 10 μm of cabozantinib. (B) 18 F-FDG uptake in K1 cells induced by 5 μm of sorafenib or 10 μm of cabozantinib. Data are expressed as mean ± SD. *, P < 0.001, compared with untreated K1 cells;, P < 0.001, compared with glucose blocked K1 cells.