Human PTC cell line K1, BCPAP and ATC cell line 8505C were purchased from the Institute of Biochemistry and Cell Biology (SIBS, CAS, Shanghai, China). BHP27 cell line was a kind gift from Professor Li-Bo Chen in our hospital. Both the cell lines used in the present study have been confirmed for identity (Fumed Biotech Bio-Medicine; SIBS, CAS). All cell lines used in these experiments were maintained in RPMI-1640 medium (cat. #11875-093) supplemented with 10% fetal bovine serum (FBS) (cat. #16000-044) (both from Gibco, Carlsbad, CA, USA) in a 5% CO₂-95% air atmosphere at 37°C.

In order to determine the half maximal inhibitory concentration (IC₅₀) of propranolol, atenolol and ICI118551, both from Sigma (St. Louis, MO, USA); ICI118551 from MedChem Express (Monmouth Junction, NJ, USA), and the effect of isoprenaline hydrochloride (Sigma) on growth, a CCK-8 assay (Yeasen, Shanghai, China) was performed. The cells were seeded into 96-well plates at a density of 1×10⁴ cells/well for 24 h, and then incubated for 24 h with increasing concentrations of the various compounds under study (10 and 40 µM for isoprenaline; 50, 100, 150, 200, 250, 300, 350 and 400 µM for propranolol, atenolol and ICI118551). Control cells were allowed to grow in the absence of any inhibitors for the same period of time. The samples were assayed in sextuplet and at least in three independent experiments, and the mean value for each experiment was calculated. Results are presented as mean (± SEM) and are expressed as percentage of the control group. For clonogenic survival studies, K1 cells were pretreated with ABT-737 (1 µM) or propranolol (200 µM) for 24 h before treatment with vemurafenib (PLX-4032) (5 µM) for 72 h, and then the media were changed and colonies were stained with crystal violet 10 days after treatment and imaged. For nuclear fragmentation assay, 8505C cells were treated with propranolol (0, 200 and 500 µM) for 24 h and the cells were stained with Hoechst 33342 and imaged by fluorescent microscopy.

Before flow cytometric apoptosis analysis, 8505C cells were pretreated for 24 h with increasing concentrations of propranolol (0, 100, 200, 300, 400 and 500 µM). At each concentration half a million cells were collected, washed 2 times with cold phosphate-buffered saline (PBS). Fixed cells were stained with Annexin V-FITC/PI (Yeasen). For cell cycle analysis, cells (10⁶) were treated with propranolol (100 and 200 µmol/l) for 0, 24 and 48 h. After the treatment, the cells were trypsinized and centrifuged at 800 rpm for 5 min. Subsequently, cells were collected and washed 2 times with PBS. Cell pellets were resuspended in 300 µl ice-cold PBS and fixed overnight by adding 700 µl ethanol. After washing with PBS, cell pellets were resuspended and incubated in 300 µl PBS (containing 20 µl Rnase A) at 37°C for 30 min. In addition, 400 µl propidium iodide (PI) solution was added and incubated at 4°C for 30–60 min in the dark. Samples were analyzed on a flow cytometer (Beckman Coulter, Brea, CA, USA).

8505C cells were plated at a density of 20×10⁴ cells/well in 6-well plates. At confluence, propranolol and ICI118551 at doses of 0, 25, 50 and 100 µM were incubated with 8505C cells for 24 h. Cells were harvested in RIPA lysis buffer containing proteinase and phosphatase inhibitors. Protein was quantified using a protein assay kit (Bicinchoninic Acid; Yeasen). Equal amounts of cell lysates were separated by 12% SDS-PAGE, and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with Tris-buffered saline (TBS) containing 5% skimmed milk powder for 1 h, and then probed with specific antibodies [anti-ADRB1 from Aviva Systems Biology Corporation (San Diego, CA, USA); anti-ADRB2, anti-Akt, anti-Bcl-2, anti-MCL1, anti-Bcl-xL, anti-Bax, anti-CCND1, anti-HK2 and anti-GAPDH from ProteinTech (Chicago, IL, USA); anti-mTOR, anti-phospho-mTOR and anti-phospho-Akt from Signalway Antibody (SAB; Signalway Antibody, College Park, MD, USA); anti-GLUT1 from novus Biologicals (Littleton, CO, USA); anti-β-actin from Sigma] overnight at 4°C and followed by horseradish peroxidase (HRP)-labeled goat anti-mouse IgG or HRP-labeled goat anti-rabbit IgG (both from Abcam, Cambridge, MA, USA) for 1 h. The membranes were developed using the enhanced plus chemiluminescence assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Images were analyzed using Image-Pro Plus 6.0.

All protocols involving mice were evaluated and approved by our Institutional Animal Care and Use Committee and performed under veterinary supervision. Nude mice (5-week-old) were injected with 2×10⁶ 8505C cells in RPMI-1640 medium subcutaneously in the left flank. When tumors reached 50 mm³ mice were injected subcutaneously with propranolol (Sigma) dissolved in PBS (n=15) or PBS alone (n=10) at a dose of 10 mg/kg/day for up to 15 days. Tumor growth was monitored every two days and tumor volume was calculated (volume = length x width²/2). Following in vivo pharmacologic intervention, 130–200 µCi ¹⁸F-FDG in 200 µl of saline were injected into the tail vein of each mouse. Anesthesia was performed with isoflurane anesthesia system. The PET/CT data acquisition procedure was performed on Siemens Inveon PET-CT when the mice were fully anesthetized. Body temperature was maintained using a heating pad equipped with the micro PET/CT system. All PET/CT images were processed and analyzed using Intrasense software. After PET/CT imaging, mice bearing ATC were sacrificed and resected tumors were weighted, followed by fixation of the specimens.

The resected tumor specimens of ATC xenografts were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were cut on a microtome and mounted on glass slides. Sections were dewaxed and hydrated in graded alcoholic solutions and then distilled water. Routine hematoxylin and eosin (H&E) staining was carried out. Immunohistochemical staining for Bcl-2, CCND1 and Ki-67 were performed using the SABC kit according to the manufacturer's instructions.

Statistical analyses were performed using the Statistical Package for the Social Sciences, version 20.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant result.

As is shown in Fig. 1A, western blotting of 8505C and K1 lysates demonstrated that ADRB 1/2 could be detected in both 8505C and K1 cell lines. ADRB2 is relatively higher expressed in 8505C cells than that expressed in K1 cells. It seems that the expressions of ADRB1 between the two cell lines are comparable. Positive expression of ADRB1/2 suggests that propranolol may influence the viability of thyroid cancer cells through targeting ADRB1/2.

To determine the effect of propranolol on thyroid cancer cells, CCK-8 assay was used to determine the half-maximal inhibitory concentration (IC₅₀). Growth curves of 8505C and K1 cells treated with increasing doses of propranolol for 24 h are shown in Fig. 1B. For 8505C and K1, the IC₅₀ was 200 and 280 µM, respectively, which were higher than that reported in neuroblastoma (5) and similar to those reported in other cancer cells (3).

Next, we explored whether β1-AR or β2-AR is involved in the inhibitory effect of propranolol, 8505C cells were treated with non-selective β-blocker propranolol, β1-specific antagonist atenolol and β2-specific antagonist ICI118551 hydrochloride. With the concentrations increased, propranolol and ICI118551 showed equivalent inhibitory function on the growth of 8505C while atenolol had no effect, suggesting that propranolol induces cell death via blocking β2-AR rather than β1-AR (Fig. 1C). In contrast to the growth suppression in response to β-blockers, we observed that isoproterenol induced a dose-dependent cell growth in both 8505C and K1 cells and this effect can be reversed by pretreatment using propranolol (50 µM) (Fig. 1D).

In order to study the mechanism of propranolol-induced inhibition on the viability of 8505C cells, we detected cell apoptosis in the normal control and propranolol-treated 8505C cells as depicted in Fig. 2. Fig. 2A–F shows that following the propranolol treatment (0–500 µM) for 24 h, FCM revealed a dose-dependent increase in Annexin V-FITC/PI-positive apoptotic cells (apoptosis rate from 0 to 500 µM: 1.8, 7.8, 13, 17.5, 27.8 and 27.6%). In addition, propranolol induced apoptosis was further characterized by nuclear fragmentation, a hallmark feature of apoptosis (Fig. 2G–I). These data suggested that propranolol was able to induce apoptosis of 8505C cells in a dose-dependent manner in vitro.

Bcl-2 family members, including Bcl-2, Mcl-1, Bcl-xL, Bim, Bax and Bak, have been reported to be involved in the mitochondrial manner of apoptosis, of which anti-apoptotic Bcl-2 is overexpressed in certain solid tumors and inhibition of Bcl-2 may enhance apoptosis and primary responses to targeted therapy (12–14). Following treatment with increasing concentrations of propranolol and ICI118551 for 24 h, Bcl-2 decreased after treatment with propranolol and ICI118551, and these two drugs yielded similar inhibitory effect in 8505C cells. Notably, expression levels of other Bcl-2 family members did not change following propranolol treatment (Fig. 3D). Given the central role of PI3K/Akt/mTOR pathway in tumorigenesis, the fact that AKT directly phosphorylates pro-apoptotic BAD and restores anti-apoptotic Bcl-xL and Bcl-2 (15–17), we next investigated the expression profiles of Akt and mTOR. Western blotting of 8505C lysates validated that propranolol and ICI118551 significantly suppressed the expression levels of p-Akt and p-mTOR in 8505C cells at a relatively higher concentration (Fig. 3C).

Furthermore, we performed cell cycle analysis in the propranolol-treated 8505C cells as shown in Fig. 3A and B. Exposure of 8505C cells to propranolol resulted in the enrichment of G0/G1 phase accompanied by a decrease in the S phase in a dose- and time-dependent manner. The cell cycle regulation protein cyclin D1, which is responsible for cell cycle progression (18), was also investigated in the present study. The expression level of cyclin D1 was dose-dependent and significantly decreased in both propranolol and ICI118551 treated groups when compared to the normal controls (Fig. 4C). Similarly Zhang et al also reported decreased expression of Bcl-2 and cyclin D1 induced by propranolol in pancreatic cancer cells (8).

During dedifferentiation process from differentiated thyroid cancer to anaplastic thyroid cancer, an inverse relationship between radioiodine and fluorodeoxyglucose uptake was observed (19). Among the major proteins regulating the transportation and metabolism of glucose, GLUT-1 and HK-2 are closely related to the rate of ¹⁸F-FDG uptake in cancers. Therefore, we investigated the relationship between the blockage of ADRB and the expression of GLUT-1 and HK-2 in 8505C cells in vitro. The results of the in vitro propranolol and ICI118551 intervention revealed that the expression of both GLUT-1 and HK-2 significantly decreased after treatment using β-blockers (Fig. 5A).

Finally we investigated the effect of propranolol on the growth of 8505C xenografts in vivo. Under the circumstances that ATC is high metabolic and the tumors in the treatment group shrinked (Fig. 5B), first we assessed the nude mouse tumors using ¹⁸F-FDG PET/CT scan. Coronal computed tomography validated that tumor of the control group was bigger than that of the propranolol treated group (Fig. 5C and D). SUV_max of the control group is statistically higher than that of the propranolol intervention group (SUV_max 8.9 vs. SUV_max 2.1). It was noteworthy that the mean body weights of the two groups were not statistically significant (data not shown), suggesting limited toxicity of propranolol in the short-term. Although no low density areas appeared on the CT scans of the control mice, H&E staining of the tumor specimens of the control group showed necrosis in the middle area of the slice (Fig. 6A), partially reflecting the aggressive property of ATC in the control group mainly caused by rapid tumor progression. Consistent with western blot results, immunohistochemistry analysis of the resected tumor tissue showed decreased cyclin D1 and Bcl-2 in the propranolol treated group when compared with normal controls (Fig. 6B). Delayed tumor proliferation in the experimental group was further supported by immunohistochemical staining of tumor cells with the proliferation marker Ki-67 (Fig. 6B).

The present study confirmed the previous observation that BRAF^V600E inhibitor vemurafenib induces cell cycle arrest of BRAF mutant cell lines K1 and BCPAP, but had little impact on the 8505C and BRAF-WT BHP27 cells (Fig. 7A and B). In addition, we found that G0/G1 cell cycle arrest was correlated with reduced phosphorylation of ERK1/2 and reduced expression of cyclin D1 in K1 cells time-dependently (Fig. 7C). However, as was seen from the colony formation assay, vemurafenib (PLX-4032) alone only inhibited the proliferation of K1 cells, but was insufficient to induce apoptosis. Neither ABT-737 (a Bcl-2 inhibitor) nor the combination of ABT-737 and vemurafenib resulted in profound synergism or extensive tumor cell death (Fig. 7D; data not shown), although ABT-737 has been proven to be potent when used along with MEK inhibitor or vemurafenib in melanoma (13,20). Notably propranolol alone induced apoptosis of K1 cells at a relatively higher concentration (Fig. 8A). Most importantly, propranolol pretreatment (200 µM) sensitized K1 cells to the cytotoxicity of vemurafenib characterized by the loss of clonogenic survival and enhanced apoptosis (Fig. 8B and C). These preliminary results demonstrated that propranolol may enhance the cytotoxicity and minimize the side-effects of the targeted molecular therapy.