Bortezomib sensitizes thyroid cancer to BRAF inhibitor in vitro and in vivo

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   Figure 1

   (A) Immunohistochemistry of PTC-bearing BRAF^V600E 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 BRAF^V600E and the thyroid cancer cell line (KAT18) expressing BRAF^wt up on the expression of BRAF^V600E and activation of NF-κB. GAPDH was used as internal control for loading and transfer.
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   Figure 2

   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.
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   Figure 3

   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 IΚBα 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.
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   Figure 4

   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.

• © 2018 Society for Endocrinology