Cell lines with 8505C harboring BRAF^V600E mutation were purchased from Deutsche Sammlung von Mikro-organismen and Zellkulturen (DSMZ; Braunschweig, Germany); HTh7 was gifts given by Dr. Minho Shong (School of Medicine, Chungnam National University). The 8505C cell line was maintained in Roswell Park Memorial Institute-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin–streptomycin (HyClone). HTh7 cell line harboring NRAS (BRAF^V600E wild-type) was maintained in Dulbecco’s modified Eagle’s medium (DMEM high glucose; HyClone) supplemented with 10% FBS and 1% penicillin–streptomycin.

Lenvatinib as a multi-kinase inhibitor and vemurafenib as a BRAF inhibitor were purchased from Selleckchem (Pittsburgh, PA, USA). A 10mM stock solution of each inhibitor was prepared by dissolving with dimethyl sulfoxide and was stored at −20°C before their usage.

Cell viability of the thyroid cancer cell lines after lenvatinib and vemurafenib treatment were determined using a cell counting kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Kumamoto, Japan). For single treatment, multiple dose of vemurafenib (0, 0.25, 0.5, 1, 2, 4, 8 μM) and lenvatinib (0, 0.78, 1.53, 3.13, 6.25, 12.5, 25 μM) were applied to 8505C and HTh7 cells. Based on single treatment results, 0, 0.25, 0.5 μM of vemurafenib and 0, 0.78, 1.53, 3.13, 6.25, 12.5, 25 μM of lenvatinib were applied to 8505C and HTh7 cells. Firstly, the cells (5×10³) were seeded in 96-well plates and incubated for 24 hours in a humidified incubator at 37°C with 5% CO₂. On the next day, various concentrations of lenvatinib and vemurafenib were applied to cells, which were incubated for an additional 72 hours in the CO₂ incubator at 37°C. After then, CCK-8 reagent was added in each well, and light absorbance at 450 nm was measured to assess the cell viability. The cell viability test was performed with quintuplicates, and the highest and the lowest values of the tests were excluded for the analysis.

The 8505C cells were exposed at fixed lenvatinib and vemurafenib concentrations for 72 hours. Following the incubation, cell pellets were collected and lysed using radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Rockford, IL, USA) containing a protease and phosphatase inhibitor cocktail kit (Thermo Fisher Scientific). Lysed cells were vortexed three times at 10 minutes intervals and then centrifuged at 13,000 g for 20 minutes at 4°C. Next, the supernatants from lysed cells was collected and quantified using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). For western blot analysis, 20 μg proteins were electrophoresed on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 3% bovine serum albumin (BSA) prepared in Tris-buffered saline containing Tween-20 (TBS-T) and then probed with primary antibodies overnight at 4°C. Next day, membranes were washed using TBS-T and probed with a horseradish peroxidase (HRP)-conjugated secondary antibody for one hour at room temperature. Membranes were then washed three times with TBS-T and signals were visualized by using an enhanced chemiluminescence detection reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Protein bands were detected using an X-ray films with imager system. PARP (Cell Signaling, Danvers, MA, USA), cleaved PARP (Cell Signaling), GAPDH (Santa Cruz, Dallas, TX, USA) antibodies were used as primary antibodies with dilution ratio of 1:5,000. Anti-mouse and anti-rabbit HRP-conjugated secondary antibody (Dilution ratio 1:10,000; Cell Signaling) was used.

After confirming the cell viability, suitable concentrations (lenvatinib 12.5 μM, vemurafenib 1 μM) was used for further experiments. The thyroid cancer cells (3×10⁴) were cultured for 72 hours in 4-well chamber slides in the humidified incubator at 37°C with 5% CO₂. For γH2A.X, as a DNA damage marker, immunofluorescence imaging, 8505C cells were treated with lenvatinib 12.5 μM or vemurafenib 1 μM alone or with a combination of both kinase inhibitors. Following incubation for 72 hours, cells were fixed with chilled methanol for 10 minutes at −20°C and washed three times with PBS at 10 minutes intervals. Cells were then permeabilized by incubating with 0.5% Triton X-100 for 90 seconds, followed by three washes with PBS at 10 minutes intervals. Cells were blocked with 3% BSA in PBS containing 0.1% Triton X-100 for one hour and were incubated an anti-γH2A.X primary antibody conjugated with Dylight 488 (Abcam) at room temperature for 4 hours. Coverslips were mounted onto chamber slides using Vecta mounting medium containing 4’,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). γH2A.X-stained cells were observed using confocal laser microscopy (LSM 5 exciter; Zeiss, Oberkochen, Germany).

All data are expressed as mean±standard deviation. The results from a Student’s t-test of the treated cells were compared with those of the control cells using Excel software (Microsoft, Redmond, WA, USA). A p-value of <0.05 was considered to be statistically significant.

To investigate the anti-proliferative effect of lenvatinib and vemurafenib, we chose two ATC cell lines with different mutations. Anti-proliferative effect of vemurafenib on 8505C BRAF^V600E-mutated cells showed from 0.25 μM concentration. However, HTh7 cells with NRAS mutation represented drug resistance up to 4 μM and abruptly cell viability was decreased from 8 μM (Fig. 1A). There were statistical differences of cell viability in 0.25 to 4 μM concentration of vemurafenib between two cell lines (p<0.05). In case of lenvatinib treatment as a multi-kinase inhibitor, 8505C cells harboring BRAF mutation gradually decreased cell viability dose-dependent manner. HTh7 harboring NRAS mutation also showed similar pattern of cell viability with 8505C cells after lenvatinib treatment until 12.5 concentration of lenvatinib (p>0.05). Only in 25 μM concentration of lenvatinib, there was statistical difference of cell viability between two cell lines (p<0.05) (Fig. 1B). These results represented that vemurafenib as a BRAF inhibitor had an effect for inhibiting of proliferation in BRAF-mutated thyroid cancers, but not in NRAS-mutated thyroid cancers.

From the results with single treatment, we selected suitable concentrations for evaluating combination treatment. Next, we compared cytotoxic effect between single and combination treatments. HTh7 cells harboring NRAS mutation have only some additive effects of vemurafenib compared with lenvatinib single treatment (Fig. 2A). There were statistical differences of cell viability in 3.13 to 6.25 μM of lenvatinib between lenvatnib only and combination of lenvatinib＋0.25 μM vemurafenib (p<0.05). And there were statistical differences of cell viability in 3.13 to 12.5 μM of lenvatinib between lenvatnib only and combination of lenvatinib＋0.5 μM vemurafenib (p<0.05).

However, 8505C cells harboring BRAF^V600E mutation showed the synergistic anti-proliferative effect with lenvatinib and vemurafenib (Fig. 2B). There were statistical differences of cell viability in 1.56 to 12.5 μM of lenvatinib between lenvatnib only and combination of lenvatinib＋0.25 μM vemurafenib (p<0.05). And there were statistical differences of cell viability in every concentration of lenvatinib between lenvatnib only and combination of lenvatinib＋0.5 μM vemurafenib (p<0.05). Interestingly, 1.56 μM concentration of lenvatinib with 0.25 μM concentrations of vemurafenib demonstrated a synergistic effect.

Based on previous results, we pursued to monitor DNA damage via lenvatinib and vemurafenib combination treatment in 8505C cells harboring BRAF^V600E mutation. To perform the immunofluorescence experiment, 12.5 μM concentration of lenvatinib and 1 μM concentration of vemurafenib were selected. Treating 8505C cells with lenvatinib alone triggered the DNA damage trivially in nucleus. However, vemurafenib single treatment induced the DNA damage sub-stantially. Moreover, combination treatment with vemurafenib and lenvatinib markedly enhanced the DNA damage suggesting the synergistic effect of the two kinase inhibitors in BRAF^V600E-mutated thyroid cancer cells (Fig. 3).

After investigating the effects of lenvatinib or/and vemurafenib on DNA damage, we evaluated the change of apoptosis induction in thyroid cancer harboring BRAF^V600E mutation. Poly (ADP-ribose) polymerase (PARP), as a nuclear enzyme, is involved in various cellular processes, including DNA repair, apoptosis. As shown Fig. 4, we confirmed both expression of PARP and cleaved-PARP after the treatments. Although total PARP expression was not changed by the treatments, cleaved PARP was higher in both lenvatinib and vemurafenib single treatments and was highest in combination treatment. These results suggested that lenvatinib and vemurafenib combination treatment could induce apoptosis strongly than the single drug treatments.