Sorafenib inhibits intracellular signaling pathways and induces cell cycle arrest and cell death in thyroid carcinoma cells irrespective of histological origin or BRAF mutational status

Abstract

Background: Patients with dedifferentiated or anaplastic thyroid carcinomas currently lack appropriate treatment options. Kinase inhibitors are among the most promising new agents as alternative strategies. The BRAF- and multi-kinase inhibitor, sorafenib, has already shown antitumor effects in thyroid carcinoma patients in a phase III clinical trial. In this study we aim to better characterize molecular effects and efficacy of sorafenib against thyroid carcinoma cells with various histological origins and different BRAF mutational status. Analysis of different signaling pathways affected by sorafenib may contribute to assist a more specific therapy choice with fewer side effects. Twelve thyroid carcinoma cell lines derived from anaplastic, follicular and papillary thyroid carcinomas with wildtype or mutationally activated BRAF were treated with sorafenib. Growth inhibition, cell cycle arrest, cell death induction and inhibition of intracellular signaling pathways were then comprehensively analyzed.

Methods: Cell viability was analyzed by MTT assay, and the cell cycle was assessed by flow cytometry after propidium iodide staining. Cell death was assessed by lactate dehydrogenase liberation assays, caspase activity assays and subG1 peak determinations. Inhibition of intracellular pathways was analyzed in dot blot and western blot analyses.

Results: Sorafenib inhibited proliferation of all thyroid carcinoma cell lines tested with IC50 values ranging between 1.85 and 4.2 μM. Cells derived from papillary carcinoma harboring the mutant BRAF (V600E) allele were slightly more sensitive to sorafenib than those harboring wildtype BRAF. Cell cycle analyses and caspase assays showed a sorafenib-dependent induction of apoptosis in all cell lines, whereas increased lactate dehydrogenase release suggested cell membrane disruption. Sorafenib treatment caused a rapid inhibition of various MAP kinases in addition to inhibiting AKT and receptor tyrosine kinases.

Conclusions: Sorafenib inhibited multiple intracellular signaling pathways in thyroid carcinoma cells, which resulted in cell cycle arrest and the initiation of apoptosis. Sorafenib was effective against all thyroid carcinoma cell lines regardless of their tumor subtype origin or BRAF status, confirming that sorafenib is therapeutically beneficial for patients with any subtype of dedifferentiated thyroid cancer. Inhibition of single intracellular targets of sorafenib in thyroid carcinoma cells may allow the development of more specific therapeutic intervention with less side effects.

Figure 1

Sorafenib reduced the viability of thyroid carcinoma cell lines of different histological derivation. Cells were cultured with increasing concentrations of sorafenib or vehicle (DMSO) control for 48 h, and viability was assessed by MTT assay. Values are reported as percent of vehicle control ± standard deviation, and represent mean values of eight determinations of one representative experiment of three. IC50 values and the lowest concentration that caused a significant loss of viability for all cell lines examined are depicted in Table 1.

Figure 2

Cell cycle changes in C643 cells before and after incubation with 3 μM sorafenib for 24 h hours. Cell cycle analysis was conducted using FACS, and this figure shows the complete results for one cell line as an example. Besides the increase in SubG1 peak, in the remaining living cells a decrease in G1 phase and in G2/M-phase and an increase in S-phase of cell cycle was observed. Values for the other cell lines examined are depicted in Table 2.

Figure 3

Sorafenib induces cell death in thyroid carcinoma cell lines. SW1736, HTh7, BHT101 and ML1 cells were incubated for 14 h and 24 h with 3 μM sorafenib or vehicle (DMSO). LDH release into the cell culture medium was measured using the Cytotox assay (a), and increased caspase 3 and 7 activity was detected using the ApoOne assay (b). Data represent mean values of eight-fold determinations ± standard deviation, and are depicted as percent of vehicle-treated control. *indicates significant increase (p<0.05, Student’s t-test).

Figure 4

Sorafenib suppressed phosphorylation of ERK, p38-MAP kinase, JNK and AKT in SW1736 and BHT101 thyroid carcinoma cells. Cells were treated with 3 μM sorafenib for 1, 5 and 10 minutes. Whole-cell lysates were examined using western blot analysis. Expression of total protein was used as control. Signal intensities of phosphorylated proteins were corrected for signal intensities of total proteins and expressed as percent of untreated control.