Journal: Molecular Endocrinology

Article Title: FOXO1 Controls Thyroid Cell Proliferation in Response to TSH and IGF-I and Is Involved in Thyroid Tumorigenesis

doi: 10.1210/me.2012-1032

Figure Lengend Snippet: FoxO1 expression is decreased in Ras-transformed FRTL5 cells, human thyroid tumor cell lines, and human thyroid tumors. A, Cells were cultured for 2 d in complete medium including thyrotropin and IGF-I, and total protein extracts were recovered and submitted to a Western blot assay to detect FoxO1 and tubulin levels as loading control. A Western blot representative of three independent experiments is shown. B, BRAFV600E PCCl3 cells were grown in 6H medium for 2 d and then cultured in the absence of thyrotropin, IGF-I, and serum for 2 more days. Then cells were treated (+) or left untreated (−) with doxycycline for increasing times. After protein extraction, FoxO1 levels were detected by Western blot. diP-ERK is shown to confirm doxycycline-induced BRAFV600E expression, and tubulin is shown as loading control. C, Total RNA from PCCl3 cells growing in the presence of thyrotropin and IGF-I from surgically removed normal thyroid tissue (N2, N3, N4, N5 and N7) and from TPC1, WRO, FRO, and SW1736 human thyroid tumor-derived cell lines was extracted, and a qRT-PCR assay was performed to detect FoxO1 mRNA. Results are the mean ± sd of FoxO1 mRNA levels relative to the β-glucoronidase (GUS) levels from three independent experiments performed in triplicate and changes respective to the levels of the differentiated cells were considered significant at P < 0.05. **, P =0.01–0.001 (two tailed t test). D, PCCl3 cells were cultured in the absence (−) or presence (6H) of thyrotropin, IGF-I, and serum for 2 d. Tumor cells were seeded and cultured for 2 d in complete medium; next, total protein extracts were recovered and submitted to a Western blot assay to detect the levels of FoxO1, pSer473Akt (pAkt), phospho-p44/42 Erk1/2 (dipERK), and tubulin as loading control. A Western blot representative of three independent experiments is shown. E, Tumoral (T) and normal (N) human thyroid tissue from the corresponding unaffected lobe was obtained from seven different patients. Total RNA was extracted and qRT-PCR was performed to detect FoxO1 mRNA levels. Results are the mean ± sd of three independent experiments performed in triplicate and changes with respect to the mRNA levels of normal cells were considered significant at P < 0.05. *, P = 0.05–0.01; **, P = 0.01–0.001 (two tailed t test).

Article Snippet: We are grateful to Dr. James Fagin (Memorial Sloan-Kettering Cancer Center, New York, NY) for WRO and BRAF-PCCl3 cells, to Dr. Erik Heldin (Rudbeck Laboratory, Uppsala, Sweden) for SW1736 cells, and to Drs. Alfredo Fusco and Roberto Di Lauro (Università degli Studi di Napoli Federico II, Naples, Italy) for TPC1 and K- and H-Ras FRTL5 cells, respectively.

Techniques: Expressing, Transformation Assay, Cell Culture, Western Blot, Control, Protein Extraction, Derivative Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Molecular Endocrinology

Article Title: FOXO1 Controls Thyroid Cell Proliferation in Response to TSH and IGF-I and Is Involved in Thyroid Tumorigenesis

doi: 10.1210/me.2012-1032

Figure Lengend Snippet: FoxO1 reexpression increases p27KIP1 promoter activity and decreases proliferation of WRO and SW1736 human thyroid tumor cells. A, WRO and SW1736 human thyroid tumor cell lines were transfected with 3 μg of the FoxO1ADA nuclear mutant or vehicle/control vector, together with 1 μg of the p27KIP1-Luc and 0.2 μg of the CMV-Renilla expression vectors to measure the activity of the p27KIP1 promoter. After 24 h transfection, cells were collected for the determination of luciferase and Renilla levels. Results are shown as the mean ± sd of the luciferase levels relative to the nonregulated Renilla levels of three independent experiments. The changes in the activity of the p27KIP1 promoter in cells transfected with the FoxO1ADA expression vector relative to cells transfected with vehicle vector were considered significant at P < 0.05. *, P = 0.05–0.01 (two tailed t test). B, WRO and SW1736 cells were transfected with 1.2 μg of the FoxO1ADA constitutive nuclear mutant or vehicle vector (control). Cells were fixed 24, 48, and 72 h later and treated for the measurement of BrdU incorporation. The results show the mean ± sd of BrdU incorporation of FoxO1ADA-expressing cells relative to the incorporation of cells transfected with the vehicle vector from three independent experiments. *, P = 0.05–0.01; **, P =0.01–0.001 (two tailed t test).

Article Snippet: We are grateful to Dr. James Fagin (Memorial Sloan-Kettering Cancer Center, New York, NY) for WRO and BRAF-PCCl3 cells, to Dr. Erik Heldin (Rudbeck Laboratory, Uppsala, Sweden) for SW1736 cells, and to Drs. Alfredo Fusco and Roberto Di Lauro (Università degli Studi di Napoli Federico II, Naples, Italy) for TPC1 and K- and H-Ras FRTL5 cells, respectively.

Techniques: Activity Assay, Transfection, Mutagenesis, Control, Plasmid Preparation, Expressing, Luciferase, Two Tailed Test, BrdU Incorporation Assay

Journal: Endocrine-related cancer

Article Title: Bortezomib sensitizes thyroid cancer to BRAF inhibitor in vitro and in vivo

doi: 10.1530/ERC-17-0182

Figure Lengend Snippet: (A) Immunohistochemistry of PTC-bearing BRAFV600E using NF-κB-, Ki67- and Cyclin-D1-specific antibodies. Advanced PTC and goiter were stained with anti-NF-κB, KI67 or Cyclin D1 as indicated in PTC (right lane) and goiter (left lane). Magnification was 40×. (B) Immunofluorescence staining of PTC with VE1 and/or NF-κB. (C) Western blot analysis of the thyroid cancer cell lines (NPA, DRO, SW1736) expressing mutated BRAFV600E and the thyroid cancer cell line (KAT18) expressing BRAFwt up on the expression of BRAFV600E and activation of NF-κB. GAPDH was used as internal control for loading and transfer.

Article Snippet: Cell culture The human anaplastic thyroid carcinoma-derived cell lines, SW1736 and KAT18 cells were provided by Dr Xing (University of Johns Hopkins, Baltimore, MD, USA).

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Western Blot, Expressing, Activation Assay, Control

Journal: Endocrine-related cancer

Article Title: Bortezomib sensitizes thyroid cancer to BRAF inhibitor in vitro and in vivo

doi: 10.1530/ERC-17-0182

Figure Lengend Snippet: Cell viability assay in TC cells. (A) DRO, SW1736 and KAT18 cells were treated with Vemurafenib (0–10 μM) and Bortezomib (0–100 nM) for 120 h, and cell viability was assayed by WST-8 assay. (B) NPA, DRO, SW1736 or KAT18 cells were exposed to sub-IC50 concentrations of Vemurafenib (0.1, 0.1, 1.0 or 10 μM) and Bortezomib (15, 15, 20 or 35 nM), respectively, for 72 h, and cell viability was tested by Trypan blue nuclear exclusion assay. B; Bortezomib; V; Vemurafenib. (C) SW1736 cells were treated with Vemurafenib (1.0 μM) and Bortezomib (20 nM) for 14 days and assessed by colonogenic assay.

Article Snippet: Cell culture The human anaplastic thyroid carcinoma-derived cell lines, SW1736 and KAT18 cells were provided by Dr Xing (University of Johns Hopkins, Baltimore, MD, USA).

Techniques: Viability Assay, Exclusion Assay

Journal: Endocrine-related cancer

Article Title: Bortezomib sensitizes thyroid cancer to BRAF inhibitor in vitro and in vivo

doi: 10.1530/ERC-17-0182

Figure Lengend Snippet: Drug combination induces apoptosis and cell cycle arrest in TC cells. (A) DRO and SW1736 cells were treated with Vemurafenib (0.1 or 1.0 μM) and Bortezomib (10 or 20 nM) for 48 h. TC cells were stained with propidium iodide and anti-Annexin V. The percentage of apoptotic cells is indicated (*P < 0.05, **P < 0.01). (B) SW1736 cells were treated with Vemurafenib and/or Bortezomib, and cell cycle progression was assessed by flow cytometric assay. (C) Western blot analysis demonstrates the phosphorylation and the corresponding degradation of IKBα and cleavage of PARP in control and treated SW1736 and DRO cells. GAPDH is used as an internal control for loading and transfer. Data are representative of three independent experiments. (D) Flow cytometry analysis of treated and control cells following the JC-1-staining shows the loss of mitochondrial membrane potential in response to the exposure of TC cells to the combination therapy. (E) Flow cytometry analysis of the reactive oxygen species (ROS) in control and treated TC cell following the incubation with DHR123 substrates. Data are representative of three independent experiments performed separately. B, Bortezomib; V, Vemurafenib.

Article Snippet: Cell culture The human anaplastic thyroid carcinoma-derived cell lines, SW1736 and KAT18 cells were provided by Dr Xing (University of Johns Hopkins, Baltimore, MD, USA).

Techniques: Staining, Flow Cytometry, Western Blot, Phospho-proteomics, Control, Membrane, Incubation

Journal: Endocrine-related cancer

Article Title: Bortezomib sensitizes thyroid cancer to BRAF inhibitor in vitro and in vivo

doi: 10.1530/ERC-17-0182

Figure Lengend Snippet: Effect of drug combination on xenograft thyroid tumors in mice. Mice were engrafted with 2 × 106 SW1736 cells and treated with Vemurafenib and/or Bortezomib. (A) At the end point after treating with the drug Vemurafenib, Bortezomib or combination (V + B), tumors resected and stained with H&E. (B) Treatment of mice with the drug combination (V + B) produced a synergistic effect (64% tumor size reduction compared to initial tumor size), while Vemurafenib (35%) or Bortezomib (26%) alone slightly increased tumor size compared to initial tumor. Data are expressed as the arithmetic mean ± the standard deviation and considered significant at P < 0.05. (C) Statistical analysis of IHC of Ki-67 in control and treated mice with Vemurafenib, Bortezomib or combination for 4 weeks. Nuclear staining was counted from at least five fields and expressed as percentage, * and ** represent significance at P < 0.05 and P < 0.01, respectively, compared with control (no drug). (D) Statistical analysis of IHC of Cyclin D1 in control and treated mice with Vemurafenib, Bortezomib or combination for 4 weeks.

Article Snippet: Cell culture The human anaplastic thyroid carcinoma-derived cell lines, SW1736 and KAT18 cells were provided by Dr Xing (University of Johns Hopkins, Baltimore, MD, USA).

Techniques: Staining, Produced, Standard Deviation, Control