Selective inhibition of V600E-mutant BRAF gene induces apoptosis in thyroid carcinoma cell lines

Abstract

Purpose: Papillary thyroid cancer (PTC) has a high incidence of BRAF^V600E mutation. The purpose of this study was to evaluate the potential relationship between thyroiditis and BRAF^V600E mutation status in patients with PTC. We investigated how a selective inhibitor of BRAF^V600E PLX4032 affects the proliferation and inflammatory cytokine levels of thyroid cancer.

Methods: Two thyroid cancer cell lines TPC1 and 8505C were treated with PLX4032, an analysis was done on cell growth, cell cycle, the degree of apoptosis, and levels of inflammatory cytokines. To identify the functional links of BRAF, we used the STRING database.

Results: Docking results illustrated PLX4032 blocked the kinase activity by exclusively binding on the serine/threonine kinase domain. STRING results indicated BRAF is functionally linked to mitogen-activated protein kinase. Both cell lines showed a dose-dependent reduction in growth rate but had a different half maximal inhibitory concentration value for PLX4032. The reaction to PLX4032 was more sensitive in the 8505C cells than in the TPC1 cells. PLX4032 induced a G2/M phase arrest in the TPC1 cells and G0/G1 in the 8505C cells. PLX4032 induced apoptosis only in the 8505C cells. With PLX4032, the TPC1 cells showed decreased levels of vascular endothelial growth factor, granulocyte-macrophage colony-stimulating factor, chemokine (C-C motif) ligand 2/monocyte chemoattractant protein 1, whereas the 8505C cells showed significantly decreased levels of IL-8, serpin E1/plasminogen activator inhibitor-1, and matrix metalloproteinase (MMP)-3.

Conclusion: PLX4032 was cytotoxic in both TPC1 and 8505C cells and induced apoptosis. In the 8505C cells, inflammatory cytokines such as IL-8 and MMP-3 were down-regulated. These findings suggest the possibility that the BRAF^V600E mutation needs to target inflammatory signaling pathways in the treatment of thyroid cancer.

Keywords: BRAFV600E; Cytokines; Thyroid neoplasms; Vemurafenib.

Fig. 1. Docking for PLX4032 in wild type BRAF and V600E mutant binding patterns of PLX4032 on BRAF. Molecular docking of PLX4032 with BRAF wild type and V600E. (A) The first panel illustrates PLX4032 docked into the hydrophobic core of wild type BRAF showing hydrogen, hydrophobic, and cation interactions. (B) The second panel indicates the best-docked confirmation of PLX4032 into BRAF^V600E along with binding interactions representation.

Fig. 2. Predicting the interaction network of BRAF using STRING (https://string-db.org). Interacting nodes are displayed in circles, obtained using STRING. Predicted functional partners of BRAF are shown, taking into consideration functional links, coexpression, colocalization, genetic interactions, pathway, physical interactions, predicted, and shared protein domains.

Fig. 3. Growth rate of cells after PLX4032 treatment. TPC1 and 8505C cells cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. (A) TPC1 cells were treated for 72 hours with 100 nM, 1 µM, and 10 µM of DMSO (CTL) or PLX4032. (B) 8505C cells were treated for 72 hours with 100 nM, 1 µM, 5 µM, and 10 µM of DMSO (CTL) or PLX4032. Relative cell survival rate is shown as percentage of survival vs. control cells after treatment with PLX4032. Values presented here are mean values and standard errors from at least 3 independent experiments. Square box indicates the IC₅₀ value of the TPC1 and 8505C cells. DMSO, dimethyl sulfoxide; CTL, cytotoxic T lymphocyte; IC₅₀, half maximal inhibitory concentration. ^*P < 0.05, ^**P < 0.01 vs. control.

Fig. 4. Cell cycle analysis after PLX4032 treatment. (A) TPC1 cells were treated for 72 hours with 10 µM of DMSO (CTL) or PLX4032. (B) 8505C cells were treated for 72 hours with 5 µM of DMSO (CTL) or PLX4032. Cell cycle was analyzed by flow cytometry after DNA staining with propidium iodide. Data represent percentage of cells at each stage of cell cycle. Data are from representative experiment (out of a total of 3 experiments). DMSO, dimethyl sulfoxide; CTL, cytotoxic T lymphocyte.

Fig. 5. Apoptosisanalysisafter PLX4032 treatment. (A) TPC1 cells were treated for 72 hours with 10 µM of DMSO (CTL) or PLX4032. (B) 8505C cells were treated for 72 hours with at 5 µM of DMSO (CTL) or PLX4032. The cell cycle was analyzed by flow cytometry after DNA staining with propidium iodide. All cells were stained with FITC-conjugated annexin V in buffer containing propidium iodide and analyzed by flow cytometry. For each treatment, percentage of viable cells is shown in lower left quadrant, which indicates low levels of annexin V and propidium iodide. Data are from representative experiment (out of a total of 3 experiments). DMSO, dimethyl sulfoxide; CTL, cytotoxic T lymphocyte; FITC, fluorescein isothiocyanate.

Fig. 6. Expression of IL-6 and IL-8 after PLX4032 treatment. (A) TPC1 cells were treated for 72 hours with 10 µM of DMSO (CTL) or PLX4032. (B) 8505C cells were treated for 72 hours with 5 µM of DMSO (CTL) or PLX4032. Density levels of IL-6 and IL-8 were measured with Bio-Plex 200 system (Bio-Rad, Hercules, CA, USA). DMSO, dimethyl sulfoxide; CTL, cytotoxic T lymphocyte. ^*P < 0.05, ^**P < 0.01 vs. control.

Fig. 7. Expression of VEGF and GM-CSF after PLX4032 treatment. (A) TPC1 cells were treated for 72 hours with 10 µM of DMSO (CTL) or PLX4032. (B) 8505C cells were treated for 72 hours with 5 µM of DMSO (CTL) or PLX4032. Density levels of VEGF and GM-CSF were measured with Bio-Plex 200 system (Bio-Rad, Hercules, CA, USA). VEGF, vascular endothelial growth factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; DMSO, dimethyl sulfoxide; CTL, cytotoxic T lymphocyte. ^*P < 0.05, ^**P < 0.01 vs. control.

Fig. 8. Expression of CCL2/MCP-1 and serpin E1/PAI-1 after PLX4032 treatment. (A) TPC1 cells were treated for 72 hours with 10 µM of DMSO (CTL) or PLX4032. (B) 8505C cells were treated for 72 hours with 5 µM of DMSO (CTL) or PLX4032. Density levels of CCL2/MCP-1 and serpin E1/PAI-1 were measured with Bio-Plex 200 system (Bio-Rad, Hercules, CA, USA). CCL2, chemokine (C-C motif) ligand 2; MCP-1, monocyte chemoattractant protein 1; PAI-1, plasminogen activator inhibitor 1; DMSO, dimethyl sulfoxide; CTL, cytotoxic T lymphocyte. ^*P < 0.05, ^**P < 0.01 vs. control.

Fig. 9. Expression of MMP-1, 2, and 3 after PLX4032 treatment. (A) TPC1 cells were treated for 72 hours with 10 µM of DMSO (CTL) or PLX4032 and PLX4720. (B) 8505C cells were treated for 72 hours with 5 µM of DMSO (CTL) or PLX4032. Density levels of MMP-1, -2, and -3 were measured with Bio-Plex 200 system (Bio-Rad, Hercules, CA, USA). MMP, matrix metalloproteinase; DMSO, dimethyl sulfoxide; CTL, cytotoxic T lymphocyte. ^*P < 0.05, ^**P < 0.01 vs. control.