Research ArticleBasic Science Investigations

Antitumor Effects of Proteasome Inhibition in Anaplastic Thyroid Carcinoma

Annette Altmann, Annette Markert, Vasileios Askoxylakis, Tilman Schöning, Ralf Jesenofsky, Michael Eisenhut and Uwe Haberkorn
Journal of Nuclear Medicine November 2012, 53 (11) 1764-1771; DOI: https://doi.org/10.2967/jnumed.111.101295

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Proliferation of SW1736 (A) and C643 (B) cells was evaluated by 3H-thymidine incorporation in presence of 0.05 mM cold thymidine into acid-insoluble cell fraction after exposure to bortezomib (10 nM, 100 nM, and 1 μM) graded for 12, 24, and 48 h. Specific activity (pmol) of each sample was calculated and normalized to 105 cells. Data represent mean values and SD of triplicate samples.

  • FIGURE 2.
    FIGURE 2.

    Transcription of p21CIP/WAF1 and p53 in SW1736 and C643 cells exposed to graded bortezomib concentrations for 24 h was determined by qPCR using p21CIP/WAF1- or p53-specific probe and GAPDH-specific probe as endogenous control. Values represent relative quantification of bortezomib-treated vs. untreated cells.

  • FIGURE 3.
    FIGURE 3.

    Determination of apoptosis in SW1736 (A) and C643 (B) cells treated with increasing bortezomib concentrations for 12, 24, and 48 h. Caspase-3 and caspase-7 activity was measured by Caspase-Glo 3/7 assay (Promega). Data evaluated by luminescence (RLU) and normalization to 105 cells represent mean values and SD of triplicate samples. Expression of TRAIL and DR5/TRAIL-R2 in SW1736 and C643 cells (C) exposed to bortezomib for 24 h was quantified by qPCR using TRAIL- and DR5/TRAIL-R2 probe and GAPDH probe as endogenous control. Values represent relative quantification of bortezomib-treated vs. untreated cells. **P ≤ 0.01.

  • FIGURE 4.
    FIGURE 4.

    Uptake of 3H-FDG in SW1736 (A) and C643 (B) cells after exposure to different doses of bortezomib for 12–48 h. ATC cell lines were incubated with 37 kBq of 3H-FDG and 0.1 mM cold FDG for 10 min, and radioactivity in lysates was quantified by scintillation counting. Specific activity (pmol) of each sample was calculated and normalized to 105 cells. Data represent mean values and SD of triplicate samples.

  • FIGURE 5.
    FIGURE 5.

    (A) Changes (in percentage of pretreatment value in same animal) in 18F-FDG uptake (maximum SUV) before and 1 and 2 d after therapy with bortezomib in animals bearing SW1736 and C643 tumors in right thigh (mean and SEM, n = 4). (B) Coronal and transaxial slices of the SW1736 tumor–bearing animal before therapy (left) and 2 d after therapy (right). *P ≤ 0.05.

  • FIGURE 6.
    FIGURE 6.

    Quantification of thyroid-specific gene expression in SW1736 (A) and C643 (B) cells after bortezomib treatment for 24 h. Values represent relative quantification of bortezomib-treated vs. untreated cells evaluated by qPCR using Pax8-, TTF-1-, NIS-, thyroglobulin-, thyroperoxidase-, and TSHr-specific probes. GAPDH was used as endogenous control.

  • FIGURE 7.
    FIGURE 7.

    Uptake of 125iodide within 1 h in SW1736 (A) and C643 (B) cells untreated or treated with indicated bortezomib concentrations for 72 h (uptake experiment). In efflux experiment, intracellular radioactivity of tumor cells was evaluated at 20 min after Na125I-containing medium had been replaced by nonradioactive medium. Counts (cpm) for each sample were normalized to 105 cells. Data represent mean values and SD of triplicate samples from representative experiment. *P ≤ 0.05. **P ≤ 0.01

Tables

  • TABLE 1

    Bortezomib Induces G2–M Phase Arrest in ATC Cell Lines

    Phase
    Cell lineConcentration (nM)G1SG2–M
    SW1736
     12 h047.97 ± 0.7836.77 ± 1.0715.20 ± 0.29
    10038.28 ± 1.4332.90 ± 1.4528.51 ± 0.42
     24 h042.83 ± 1.534.77 ± 0.3522.31 ± 1.02
    10034.94 ± 2.0238.68 ± 3.3526.38 ± 1.33
     48 h050.16 ± 1.4231.44 ± 1.418.40 ± 0.02
    10048.25 ± 0.7823.42 ± 0.7528.33 ± 0.03
    C643
     12 h049.57 ± 0.0331.25 ± 0.2119.17 ± 0.18
    10027.36 ± 0.1333.70 ± 1.2938.94 ± 1.42
     24 h035.90 ± 1.0640.71 ± 0.5923.39 ± 1.66
    10022.14 ± 0.0333.97 ± 0.3443.89 ± 0.37
     48 h058.27 ± 1.9429.32 ± 0.3912.41 ± 1.55
    10046.09 ± 0.7227.50 ± 1.3326.41 ± 0.35

    • Cell cycle analysis of SW1736 and C643 cells incubated without (0 nM) or with bortezomib (100 nM) for indicated times was performed using fluorescence-activated cell sorting analysis. Data represent percentage of cells in G1, S, and G2–M phases, indicated as mean values of triplicate samples and SD.

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