Background
Thyroid carcinoma originating from thyroid follicular cell is the most common endocrine malignancy [
1,
2]. About 90% of thyroid carcinomas are well differentiated, while 10% or less are poorly differentiated or anaplastic subtypes [
2,
3]. Of the differentiated carcinomas, 85 to 90% are papillary and 10 to 15% follicular subtypes. Most differentiated carcinomas progress slowly, and patients usually become disease-free after initial treatment with thyroidectomy and radioiodine ablation. In contrast, 10 to 15% of patients initially diagnosed with differentiated carcinomas experience recurrent disease [
1,
4,
5]. A reduction in radioiodine uptake and storage accompanies tumor dedifferentiation. Dedifferentiated tumors are more aggressive and lead to a worse patient outcome [
3,
5,
6]. Tumors initially categorized as poorly differentiated (PDTC) or anaplastic thyroid carcinomas (ATC) share these features early on. Anaplastic (undifferentiated) thyroid carcinomas are highly aggressive and lethal tumors that have completely lost the ability to take up iodine [
7]. Beside their aggressive growth particularly the loss of capacity to uptake iodine makes both dedifferentiated and anaplastic thyroid carcinomas difficult to treat, and confer the poor patient prognosis. Moreover, chemotherapeutic treatment proved to be largely ineffective against aggressive thyroid carcinomas [
8]. These inadequacies of current treatment protocols for dedifferentiated and anaplastic thyroid carcinomas strongly emphasize the urgent need to establish novel targeted treatment options.
A better understanding of the molecular alterations driving thyroid tumorigenesis can drive development of appropriate targeting agents for thyroid carcinoma. Mutations in genes encoding the proteins of the mitogen activated protein (MAP) kinase signaling cascade (RAS-RAF-mitogen-activated protein kinase kinase (MEK)- extracellular-signal regulated kinase (ERK)) frequently occur in thyroid carcinomas [
2,
3]. About 50% of papillary thyroid carcinomas (PTC) harbor activating mutations in the
BRAF gene (mostly
BRAF
V600E
), an effector of MEK that in turn activates the ERK1 and ERK2 mitogen-activated protein kinases (Review [
9,
10]).
BRAF mutations also occur in up to 13% of PDTCs and 35% of ATCs [
11], but in these subtypes are restricted to tumors with a papillary component or supposed to be derived from PTC [
12]. The
BRAF
V600E
mutation has been associated with advanced clinical stage, loss of iodine accumulation and has an independent prognostic value for PTC recurrence [
13,
14]. Mutations in the three
RAS genes,
HRAS, KRAS and
NRAS, have been described in all thyroid epithelial carcinoma subtypes (Review [
3]). Besides direct mutational activation of the RAS-RAF-MEK-ERK signaling pathways, receptors with intrinsic tyrosine kinase activity can also stimulate this cascade. Overexpression and autocrine activation of the epidermal growth factor receptor (EGFR) in thyroid carcinomas contributes to the activation of the RAS-MAP kinase cascade [
15,
16]. Expression of the platelet-derived growth factor receptors (PDGFR) and their ligands in undifferentiated thyroid cells [
17,
18] also activates this cascade. An aberrant activation of the RAS-RAF-MEK-ERK signaling cascade, therefore, is common in all thyroid carcinoma subtypes, and may provide targets for appropriate molecular therapies.
Inappropriate activation of the MEK-ERK kinase cascade leads to deregulated cell proliferation, dedifferentiation and improved cell survival in a variety of tumor cell types [
19]. The importance of this pathway and its frequent deregulation and mutational activation in cancers has led to development of small molecule inhibitors. One of these inhibitors is sorafenib (Nexavar®, BAY43-9006), which was originally designed to inhibit the ARAF, BRAF and RAF1 kinases [
20]. Sorafenib competitively inhibits ATP binding to RAF catalytic domains, thus, inhibiting kinase activity via stabilization of the conserved kinase domain in the inactive configuration [
21]. Sorafenib was shown to potently inhibit RAF1 kinase, wildtype BRAF and oncogenic BRAF
V600E in vitro [
22]. Moreover, sorafenib directly blocks the autophosphorylation and activation of several receptor tyrosine kinases, including PDGFRB, fibroblast growth factor receptor 1 and vascular endothelial growth factor receptors (VEGFRs) [
20]. Sorafenib decreases ERK activation in human tumor cells, inhibits cell proliferation in vitro and inhibits growth of human tumor xenografts in nude mice [
20,
23,
24]. Sorafenib has been shown to inhibit RAF activation, phosphorylation of members of the MEK-ERK kinase family and proliferation of cell lines derived from PTC and ATC harboring an activating
BRAF mutation [
25]. These effects were similar after BRAF knockdown using siRNA, suggesting a central role for mutationally activated BRAF [
25]. Furthermore, Carlomago et al. [
26] showed that sorafenib inhibits RET kinase and thus proliferation of papillary and medullary thyroid carcinoma cells harboring an oncogenic RET kinase. Sorafenib treatment inhibited proliferation and improved survival of mice with ATC xenografts [
27]. Taken together, these results demonstrate the efficacy of sorafenib against various cell lines derived from PTCs and ATCs. However, current published reports include no data directly comparing cell lines with and without
BRAF mutations or describing the effects of sorafenib in cell lines derived from follicular thyroid carcinomas (FTC).
Some clinical phase II trials and clinical studies in patients with metastatic differentiated thyroid carcinomas have shown promising results for sorafenib [
28-
32]. The majority of these studies detected no differences in treatment efficacy between thyroid carcinoma subtypes, although the low case numbers in these studies may have hindered subgroup analysis. Positive effects were reported in one phase II trial in patients with advanced ATC, which showed partial responses in 2 of 20 patients and stable disease in 5 of 20 patients [
33]. A recently published phase III multicenter, double-blind randomized and placebo-controlled trial evaluating the efficacy of sorafenib in thyroid cancer patients (DECISION study) [
34,
35] demonstrated that sorafenib significantly improved progression-free survival compared with placebo in patients with progressive radioiodine-refractory differentiated thyroid cancer independent of the clinical and genetic subgroup. Overall, sorafenib has exhibited significant antitumor activity and clinical benefits in patients with progressive and advanced thyroid carcinoma and thus is a treatment option for patients with locally recurrent or metastatic, progressive, differentiated thyroid carcinoma refractory to radioactive iodine treatment.
Since sorafenib as a multikinase inhibitor blocks various intracellular signaling pathways, significant side effects have also been reported in clinical trials [
36]. A broader analysis of the signaling molecules affected by sorafenib treatment in specific tumor cell types may thus be useful to identify cell-specific key signaling molecules for more directly targeted treatment approaches. No data are currently available on the intracellular effects of sorafenib in thyroid carcinoma cells or potential differences in sorafenib action in thyroid carcinoma cells of the papillary (with or without the
BRAF
V600E
mutation), follicular or anaplastic subtypes. The aim of the present study was to elucidate the effects of sorafenib treatment on proliferation, cell death induction and intracellular signaling pathways in various thyroid carcinoma cell lines.
Methods
Compounds and antibodies
Sorafenib (BAY 43–9006, Nexavar®) was provided by Bayer Health Care (Wuppertal, Germany), stored in 10 mM aliquots in DMSO at −20°C and further diluted in the appropriate medium. Antibodies to detect both total protein and activated phosphorylated forms of c-Jun N-terminal kinase (JNK), AKT, p44/42 MAP kinase (ERK1/2) and p38 MAPK were purchased from Cell Signaling Technology (Danvers, MA, USA).
Cell lines and cell culture
Cell lines derived from the anaplastic, papillary and follicular thyroid cancer subtypes were used in this study. The SW1736 [
37], HTh7 [
38], HTh74 [
39], HTh83 [
40], and C643 [
17] cell lines were derived from ATC. BHT101 [
41], B-CPAP [
42], and TPC [
43] cell lines were derived from PTC. ML1 [
44] and TT2609 [
45] are FTC-derived cell lines. The FTC133, FTC236 and FTC238 [
46] cell lines were derived from a single primary FTC, a lymph node metastasis and a lung metastasis from the same patient, respectively. The HTh7, HTh74, HTh83, C643 and SW1736 cell lines were a gift from Prof. Heldin (Uppsala, Sweden), and all other cell lines were purchased from ATCC (Manassas, VA, USA), ECACC (Salisbury, UK) and DSMZ (Braunschweig, Germany). Cell lines were maintained in their appropriate media supplemented with 10% fetal bovine serum (FBS, Life Technologies, Paisley, PA, USA) at 37°C at 5% CO
2.
DNA extraction and mutation analysis
Genomic DNA was isolated from cell lines using the QIAamp DNA kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Primers used to amplify exon 15 of the
BRAF gene were described elsewhere [
47]. For PCR amplification, 5 μl of DNA solution containing 200 ng DNA was used in a 50 μl reaction containing 1xPCR buffer, 1.5 mM MgCl
2, 1.5U HotMaster Taq polymerase (Eppendorf, Hamburg, Germany) and 300 nM each of forward and reverse primers. Cycling conditions were 40 cycles of 94°C for 20 sec, 55°C for 10 sec, 65°C for 35 sec. PCR products were analyzed on 3% agarose gels and purified using the QIA quick removal kit (Qiagen). Sequencing was performed using the ABI Prism BigDye Terminator Cycle sequencing kit v1.1 on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were compared to the wildtype sequences using the Sequencher software (Gene Codes, Ann Arbor, MI, USA).
Cell proliferation studies
For proliferation assays, 1 × 104 to 5 × 104 cells (cell line dependent) were seeded into 96-well plates containing the appropriate growth medium. Medium was replaced after 24 hours with culture medium without FBS but containing 0.1% bovine serum albumin (BSA) and the indicated sorafenib concentrations was added. After 48 hours, viable cells were stained with the Cell Titer Aqueous One Solution assay (Promega, Madison, WI, USA), and optical density at 490 nm was measured using an Emax microplate photometer (Molecular Devices, Sunnyvale, CA, USA). Control values without sorafenib treatment were performed as 22-fold determinations, while all concentrations of sorafenib were tested in 8-fold. Calculation of results and Student’s t-test were performed using SoftMax pro software (Molecular Devices), and IC50 values were calculated using Sigma Plot software (Systat, San Jose, CA, USA).
Determination of lactate dehydrogenase release and caspase 3/7 activity measurement
Release of lactate dehydrogenase (LDH) from cells with damaged membranes was measured by the CytoTox-ONE homogeneous membrane integrity assay (Promega). Activity of caspases 3 and 7 was measured by the Apo-ONE homogeneous Caspase 3/7 assay (Promega). 1 × 104 to 5 × 104 cells (cell line dependent) were seeded into black, transparent-bottomed 96-well plates containing the appropriate growth medium. Medium was removed after 24 h and 100 μl culture medium without FBS, but containing 0.1% BSA and the denoted sorafenib concentration, was added to each well. After 14 or 24 hours, 50 μl of medium from each well was transferred to a fresh black 96-well plate and equilibrated to 20°C. According to the manufacturer’s instructions, 50 μl of CytoTox reagent was added and reactions were incubated for 10 min in the dark. After adding 25 μl of stop solution, fluorescence was determined with excitation and emission wavelengths of 560 nm and 590 nm, respectively. Wells containing no cells, as the zero setting, and fully lysed cells, as the maximum LDH release control, were included in each experiment. Caspase 3 and 7 activity in treated cells was determined in the original stimulation plate by adding 50 μl of Apo-ONE reagent that contained a fluorometric substrate in cell lysis and reagent buffer. After 60 min, fluorescence was measured at 521 nm after excitation with 499 nm. All values were performed as 8-fold determinations. Calculation of results and Student’s t-tests were performed using SoftMax pro software (Molecular Devices).
Cell cycle analysis
Cells were plated at 1 × 105 to 5 × 105 cells/well in 6-well plates in appropriate growth medium for cell cycle analyses. Medium was replaced with medium without FBS but containing 0.1% BSA and 3 μM sorafenib 24 h later and cells were treated for the indicated times. Treated cells were harvested and fixed in cold 70% ethanol. RNase A (60 μg/ml) and propidium iodide (25 μg/ml) in PBS were added, and samples were incubated 20 minutes in the dark at room temperature. Samples were measured on a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA), and cell cycle stages were analyzed using the ModFit Software (Verity Software House, Topsham, ME, USA).
Proteome Profiler™ array and western blot analysis
Proteome Profiler™ antibody arrays (R&D systems, Mineapolis, MN, USA) and western blotting were used to assess inhibitory effects of sorafenib on intracellular signaling proteins and receptor tyrosine kinases. Cells were plated in 10 cm culture dishes, and grown for 1–2 days to 85 to 90% confluency. Medium was removed and cells were washed once and maintained in prewarmed HBSS buffer (Life Technologies) for 20 minutes before adding 3 μM sorafenib. Treated cells were washed with ice-cold PBS, and all further steps were performed on ice. Cells were lysed in lysis buffer containing cOmplete protease inhibitor and phosSTOP phosphatase inhibitor cocktails (Roche Applied Science, Mannheim, Germany). Lysates were clarified by centrifugation at 10,000 × g for 10 min at 4°C, protein concentration determined by modified Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) and 500 μg of protein from each lysate were used in dot blot analysis according to the manufacturer’s instructions. For western blotting, 30 μg of total protein was denatured by boiling for 5 minutes in SDS sample buffer, then separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories). After blocking with 5% skim milk powder or 5% BSA in TBS, blots were incubated with the appropriate primary antibody in TBS buffer containing 0.1% Triton X-100 (TBS-T) overnight at 4°C. After washing, an appropriate secondary antibody coupled to horseradish peroxidase in TBS-T was added. Bound antigens on western and dot blots were detected using the ECL Advance chemiluminescence detection kit (GE Healthcare, Piscataway, NJ, USA). Signal intensity was evaluated with a CCD camera system, and differences were calculated with the Quantity One software (Bio-Rad Laboratories).
Statistical analysis
Statistical analysis of treatment versus control groups was performed by means of the unpaired Student’s t-test using SPSS (IBM Inc, Armonk, NY, USA) or the other software packages indicated above. P-values < 0.05 were considered statistically significant.
Discussion
Here we present a detailed analysis of kinase inhibition, effects on the cell cycle and apoptosis induction by the BRAF- and multikinase inhibitor, sorafenib, in thyroid carcinoma cell lines of various histological subtypes with and without activating
BRAF
V600E
mutations. The effects of sorafenib on various intracellular signaling molecules were studied to evaluate more specific treatment options in patients with dedifferentiated thyroid carcinomas patients. We assessed the
BRAF
V600E
mutational status for all 12 cell lines used in this study. The activating
BRAF
V600E
mutation was only detected in two of the three papillary cell lines (BHT101 and B-CPAP) and in one of the four cell lines (SW1736) as previously detected and reported [
12,
48]. The HTh7, C643 and HTh83 anaplastic cell lines, the TPC papillary cell line and the FTC133, FTC236, FTC238, ML1 and TT2609 follicular cell lines harbored only wildtype alleles for
BRAF. These findings fit well with experimental and pathological evidence indicating an involvement of
BRAF mutation in the pathogenesis of about 50% of PTCs and the progression of PTC to ATC, but no occurrence of
BRAF mutations in FTC [
9,
10,
12,
49].
Proliferation of all cell lines was inhibited by sorafenib within the 48 h treatment period. To our knowledge, ours is the first report about the inhibitory effects of sorafenib not only on cell lines derived from PTCs and ATCs, but also from FTCs. It is in good agreement with recent clinical findings in the phase III DECISION trial of sorafenib in patients with iodine-refractory thyroid cancer, where positive effects of sorafenib on progression-free survival was found in all clinical and genetic biomarker subgroups [
35]. In contrast, Kloos et al. [
29] reported better clinical responses to sorafenib in patients with PTC than in those with FTC (in patients with PTC partial response in 15% and stable disease in approx. 65% of patients, in patients with FTC no partial response and stable disease in 80% of patients, in patients with ATC stable disease in 25% of patients) [
29]. IC50 values for sorafenib in the various cell lines investigated in the present study ranged from 1.85 μM to 4.2 μM, which correspond to the lower range of achievable plasma levels. A daily dose of 400 mg sorafenib administered orally or 2 doses of 200 mg per day resulted in mean plasma levels of 20 μM in patients during a phase I trial [
50]. The two papillary cell lines BCPAP and BHT101 with the
BRAF
V600E
mutations had the lowest IC50 values for sorafenib, while a slightly higher IC50 value was calculated for the TPC1 papillary cell line, which harbors no
BRAF
V600E
mutation, but the RET/PTC1 rearrangement [
51]. Cell lines derived from FTCs and ATCs responded similarly in this study, as evidenced by IC50 values within a relatively narrow range. These IC50 values for FTC and ATC cell lines were slightly higher than those for PTC cell lines, but still comparable to the lower range of plasma concentrations that are achieved in patients [
50]. Recently, Cohen et al. reported on a synergistic effect of sorafenib treatment with withaferin A in the B-CPAP and SW1736 thyroid carcinoma cell lines [
52]. IC50 values for sorafenib treatment alone were 6.3 μM (B-CPAP) and 7.6 μM (SW1736). Although the IC50 values we report are somewhat lower, with 1.85 μM for B-CPAP and 3.25 μM for SW1736, they are in the same order of magnitude with BCPAP being the more sensitive cell line. The IC50 values we report are close to the IC50 values in the 1 μM-range reported by Salvatore et al. for sorafenib treatment of the FRO, ARO, KAT4 and NPA ATC cell lines harboring
BRAF
V600E
mutations [
25]. IC50 values for thyroid carcinoma cell lines are also very close to those reported in the literature for hepatocellular carcinoma cell lines (4.5 and 6.3 μM) [
24] and melanoma cell lines (~5 μM) [
53,
54] treated with sorafenib.
The lowest sorafenib concentration that led to a significant antiproliferative effect in our study was the lowest in three cell lines without BRAF
V600E
mutations: In the fast growing C643 and FTC238 cells, significant effects on cell number were achieved with 0.01 μM. In papillary TPC1 cells significant effects were achieved with 0.05 μM sorafenib while in BHT101 and B-CPAP cells significant inhibition was achieved with 1.0 μM as the lowest concentration. The molecular reasons for these effects are unclear, and may stem from the multikinase-inhibitor activity of sorafenib. It also points to positive effects that may be achieved by sorafenib even in low concentrations due to side effects during sorafenib treatment.
Sorafenib induced cell death in all 12 thyroid carcinoma cell lines investigated here, regardless of histological derivation or the presence of the activating
BRAF
V600E
mutation. We detected increases in the percentage of cells in subG1 for all cell lines, and different influences on cell cycle progression depending on the cell line. Sorafenib induced a larger proportion of cells of papillary and anaplastic cell lines to enter subG1 than of follicular cell lines, indicating that sorafenib has a different kind of intracellular effect on DNA fragmentation in follicular cell lines. Analysis of the proportion of the treated culture that did not enter subG1 revealed that G1 arrest was induced in all PTC and two of four ATC cell lines, while S phase arrest with G1 decrease was induced in one ATC and three FTC cell lines. The TT2609 follicular cell line showed no alteration in cell cycle phases of the living proportion of the culture. These data concerning the G1 arrest together with the occurrence of a subG1 peak are in agreement with literature data on the TPC1 papillary thyroid carcinoma cell line, the TT medullary thyroid carcinoma cell line [
26] and the ARO anaplastic thyroid carcinoma cell line treated with sorafenib concentrations in a similar concentration range as we used in vitro [
25]. On the other hand, Liu et al. observed a decrease in the number of cells in G1 and an increase of cells in S phase in HepG2 hepatocellular carcinoma cells treated with sorafenib [
24]. These results indicate that sorafenib affects the cell cycle differently depending on the cellular background. Since all papillary cell lines we examined as well as the SW1736 anaplastic cell line harboring the activating
BRAF
V600E
mutation and HTh7 cells arrested in G1 after sorafenib treatment, one may speculate that inhibition of the overactivated RAF-MAP kinase pathway in these cells contributes to the G1 arrest while other, yet unidentified, molecular effects lead to arrest in the S or G2/M phases in the other cell lines.
We further characterized the mechanism of cell death in detail in four thyroid carcinoma cell lines. We chose BHT101 as an example of a PTC cell line with a heterozygous
BRAF
V600E
mutation, ML1 as a FTC cell line, SW1736 as an ATC cell line harboring the
BRAF
V600E
mutation and HTh7 as an ATC cell line with wildtype
BRAF. All four cell lines showed marked LDH release into the medium after 14 and 24 hours of treatment, confirming plasma membrane breakdown and release of cytoplasmic contents. Apoptotic cell death was confirmed by the increased activity of caspases 3 and 7 in all four cells lines. Interestingly, values for LDH release and caspase activities were in the same magnitude in all four cell lines. LDH release was slightly, but not significantly higher in SW1736 and ML1 cells compared to BHT101 and HTh7 cells. In contrast, caspase 3 and 7 activities were slightly, but not significantly elevated in
BRAF
V600E
mutation-positive SW1736 and BHT101 cells compared to HTh7 and ML1 cells. These results are in contrast to recently reported results by Preto and coworkers [
55], who reported that sorafenib treatment only significantly induced apoptosis in anaplastic thyroid cells harboring a homozygous
BRAF
V600E
mutation (8505C cell line), but not in thyroid carcinoma cells with wildtype
BRAF (C643 and TPC1 cell lines). Preto et al. used the TUNEL assay to quantify apoptosis, and since TUNEL detects DNA fragments directly, it corresponds methodically to quantification of the subG1 peaks in our study. Kim et al. [
27] on the other hand observed no correlation between the inhibition of cell proliferation or apoptotic induction (measured as subG1 peak) and the presence of the activating
BRAF
V600E
mutation in five anaplastic thyroid carcinoma cell lines treated with sorafenib [
27] which is in accordance with our results. Analysis of caspase activity, however, reflects other mechanisms of cellular death than investigation of DNA fragmentation by subG1 peak analysis and the TUNEL assay. Caspases are key effector proteins in apoptosis that initiate systemic structural disassembly in dying cells and have a multitude of intracellular substrates (Review [
56]). Concerning the effects of the
BRAF
V600E
mutation to apoptosis resistance, Lee et al. recently showed in the nontransformed PCCl3 rat thyroid cells and in the cervical carcinoma cell line, HeLa, that transfection with an inducible
BRAF
V600E
construct mediates resistance to mitochondrial-induced apoptosis following sorafenib treatment [
57]. Overall, the effect of
BRAF
V600E
mutation on apoptosis induction appears to be different in various cellular contexts. In our experimental setting, apoptosis induction and membrane disruption after sorafenib treatment was not significantly influenced by the histological origin of and BRAF mutational status of thyroid carcinoma cells.
We also examined the of sorafenib on phosphorylation of specific tyrosine kinase receptors in selected thyroid carcinoma cell lines to better assess the impact of differing cellular backgrounds from histological derivation and the presence of the activating
BRAF
V600E
mutation. Screening of receptor tyrosine kinase receptor activation to identify the inhibitory mechanism of sorafenib exhibited similar results in all four cell lines. Sorafenib inhibited phosphorylation of VEGFRs and PDGFRs receptors, but did not affect phosphorylation of insulin receptors, IGF1R and the EGF family of receptors in thyroid carcinoma cells. These results fit well with results reported for other cell types [
22,
58]. Sorafenib treatment in vivo has been shown to also inhibit these tyrosine kinase receptors in endothelial cells and, thus, be capable of inhibiting tumor vascularization [
20]. In vitro biochemical assay showed that sorafenib directly inhibits the RAF1, BRAF and oncogenic BRAF
V600E kinases, but has no significant inhibitory effects on MEK, ERK, AKT and other signaling pathways [
22]. Complex crosstalk mechanisms can occur between signaling pathways in the cell, however, that lead to stepwise activation of different pathways depending on the cellular context. The ERK kinase inhibition we observed and that has been described by others in thyroid carcinoma cells [
24-
26] can easily be explained by inhibition of receptor tyrosine kinases or the RAF1 or BRAF molecule in the RAF-MEK-ERK kinase cascade [
19]. We observed a more pronounced inhibition of ERK1 than ERK2 by sorafenib in thyroid carcinoma cells. We can only speculate about the molecular mechanism behind this effect at this time. Differential regulation of ERK1 and ERK2 was already described in other systems (Review [
59]). The diminished AKT phosphorylation we observed in thyroid carcinoma cells, beside possible direct effects, may also be the result of receptor tyrosine kinase inhibition by sorafenib and has recently been described in prostate cancer cells [
60]. Inhibition of p38 MAP kinase by sorafenib has already been reported in cell-free kinase assays [
20]. An inhibition of p38 MAP kinase and JNK by sorafenib comparable to that in our cells was also reported in human hepatoma cell lines [
61]. JNK phosphorylation was also reported to be suppressed in endothelial cells after sorafenib treatment [
62].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
M. B.-P. drafted the project design, planned and conducted experiments, analyzed the data and wrote the text. S.M. contributed to project design and discussed the data and text. M.B. conducted experiments and analyzed data. K. Worm conducted sequencing and discussed the text. K.W.S. and D.F. contributed to text writing and discussion. K.M. contributed to project design, text writing and discussion of the data and text. All authors read and approved the final manuscript.