In total, 57 head and neck cancer tissue specimens were obtained from surgically diagnosed patients (median age, 65 years; age range, 33–89 years) by the Department of Pathology in CHA Bundang Medical Center between January 2009 and December 2018. The institutional review board of the CHA Medical Center reviewed and approved our study (IRB approval no. NON2020-003-002). Clinical staging was based on the TNM staging system of the Union for International Cancer Control, 8th edition (22). The specimens were fixed in 10% neural buffered formalin for 24 h at 4°C and embedded in paraffin. Tissue blocks were divided into 3-µm sections for hematoxylin and eosin staining and immunohistochemistry of β2-AR. All cases were diagnosed as squamous cell carcinoma. Patients with neuroendocrine cancer, sarcoma or preoperative radiotherapy/chemotherapy were excluded. Tissue microarray sections were dewaxed in xylene, rehydrated in alcohol and immersed in 3% hydrogen peroxide for 40 min at room temperature. Antigens were retrieved by heating (100°C) each section in 10 mmol/l of sodium citrate buffer for 10 min. After being rinsed thrice in phosphate-buffered saline (PBS), each section was incubated with rabbit polyclonal antibodies to β2-AR (cat. no. ab61778, 1:100; Abcam) for 18 h at 4°C. Then, they were diluted and washed thrice in PBS and incubated with horseradish peroxidase (HRP)-labeled rabbit anti-mouse immunoglobulin for 30 min at room temperature. Subsequently, samples were washed thrice and stained with diaminobenzidine solution for 5 min at room temperature to visualize peroxidase activity. For negative controls, primary antibodies were replaced with PBS. For the positive control, normal upper eyelid muscles for 3 patients underwent upper eyelid surgery for ptosis correction were used. They were 3 females (median age, 58; range, 51–69). The tissue blocks were stored by the Department of Pathology in CHA Bundang Medical Center for other research purposes after IRB approval. The expression of β2-AR protein was assessed by cytoplasmic staining of the cancer cells. To stratify β2-AR expression, the immunoreactive score (IRS) system was used because of marked intratumoral heterogeneous expression in proportion and intensity in cancer tissues. The IRS system is calculated by multiplying the percentage of positive cells (0, no positive cells; 1, <10% of positive cells; 2, 10–50%; 3, 51–80%; 4, >80%) and the intensity of the staining (0, no color reaction; 1, mild reaction; 2, moderate reaction; 3, intense reaction). Thus, the IRS result is between 0 (no staining) and 12 (maximum staining), categorized into negative (0–1), mild (2–3), moderate (4–8), and strongly positive (9–12,23). A light microscope (Olympus System Microscope Model BX43; magnification, ×200 and ×400) was used to obtain images. The intensity of the staining was determined by using QuPath version 2.0 freeware (24). It was measured as the optical density (OD). The staining vectors for all images were estimated automatically before OD measurement. The samples with OD <0.2 were considered negative (score, 0), OD ranking 0.2–0.4 was considered mild (score, 1), OD ranking 0.4–0.6 was considered moderate and OD >0.6 was considered intense (score, 3).

DNA was extracted from the specimens using a DNA isolation kit (ISOLATE II Genomic DNA kit; Meridian Bioscience, Inc.). HPV 16/18- deoxyribonucleic acid (DNA) was amplified by polymerase chain reaction (PCR) and nested PCR assays using two pairs of primers to identify HPV16/18 and the HPV16/18 regions of the L1 gene and DNA polymerase (DreamTaq Green; Thermo Fisher Scientific, Inc.). The PCR primer sequences used for each gene were as follows: HPV 16 forward, 5′-ACCCAGTATAGCTGACAGT-3′ and reverse, 5′-CTCGTTTATAATGTCTACACA-3′; and HPV 18 forward, 5′-ATAGCAATTTTGATTTGTC-3′ and reverse, 5′-AAACTCATTCCAAAATATG-3′. During the first PCR, the amplification process included 35 cycles with pre-denaturation at 95°C for 4 min, denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, first elongation at 72°C for 1 min and 30 sec and additional elongation at 75°C for 10 min. Using the same reagents, the second PCR was performed on the first PCR aliquots. Nested PCR results were analyzed by 2% agarose gel electrophoresis. During the second PCR, the amplification process was conducted with initial denaturation at 95°C for 4 min, denaturation at 94°C for 30 sec, annealing at 56.4°C for 30 sec, first elongation at 75°C for 1 min and 30 sec and additional elongation at 72°C for 10 min. DNA visualization was conducted using machine electrophoresis on the Gel Doc EZ System (Bio-Rad Laboratories, Inc.).

The following four different head and neck cancer cell lines (HNCCLs) were investigated: oral cavity (YD-10B, tongue), larynx (SNU-1066, glottis), pharynx (SNU-1041) and nasal cavity (RPMI-2650) cancers. They were purchased from the Korean Cell Line Bank and were maintained in a mixture of Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) and Ham's nutrient mixture F12 (Thermo Fisher Scientific, Inc.) at a 3:1 ratio. They were supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and 50 U/ml concentration of penicillin at 37°C in 5% CO₂.

Total RNA was isolated using TRIzol reagent (Ambion; Thermo Fisher Scientific, Inc.). For reverse transcription, the AccuPower^® RT premix cDNA synthesis kit was used according to the manufacturer's instructions (Bioneer Corporation). For RT-PCR, 1 µg cDNA products were amplified using SYBR Green Supermix (Bio-Rad Laboratories, Inc.). For SYBR Green assays, β2-AR and RPS18 primers (for 18S rRNA) were used. The PCR primer sequences used for each gene were as follows: β2-AR forward, 5′-TTAGCCAGGTGGAGCAGGATG-3′ and reverse, 5′-GCCTAACGTCTTGAGGGCTTTG-3′; and RPS18 forward, 5′-GCAGAATCCACGCCAGTACAAG-3′ and reverse, 5′-GCTTGTTGTCCAGACCATTGGC-3′. Fluorescence was detected with the CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). All values were standardized using the 2^−∆ΔCq method (25). Moreover, the level of β2-AR messenger RNA (mRNA) expression was normalized to 18S rRNA. PCR conditions were: Initial activation at 95°C for 5 min, denaturation at 94°C for 40 sec, annealing at 60°C for 30 sec, 35 cycles of extension at 72°C for 60 sec and final elongation at 72°C for 8 min.

Cell lysates were isolated by adding PRO-PREP™ protein extraction solution (Intron Biotechnology, Inc.) containing various protease inhibitors, such as phenylmethylsulfonyl fluoride, ethylenediamine tetraacetic acid, pepstatin A, leupeptin and aprotinin. Total protein concentration was determined by using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Furthermore, 40 µg of protein/lane was resolved using 10% sodium dodecyl sulfate-poly acrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. The nitrocellulose membranes were blocked in 5% milk/Tris-buffered saline with 0.1% Tween-20 (TBS-T) at 25°C for 1 h and then incubated with β2-AR primary antibodies overnight at 4°C. The primary antibody was the anti-β2-AR rabbit monoclonal antibody (cat. no. ab182136; Abcam) at a 1:1,000 dilution. After being washed in TBS-T buffer, the membrane was incubated with HRP-conjugated secondary antibodies for 1 h. The secondary antibody was goat anti-rabbit IgG (cat. no. ab97051; Abcam) at 1:20,000 dilution. The internal control used was β-actin. The primary and secondary antibody used for β-actin were as follows: anti-β-actin mouse antibody (cat. no. sc-47778; Santa Cruz Biotechnology, Inc.), goat anti-mouse IgG antibody conjugated to HRP (cat. no. GTX213111-01; GeneTex, Inc.). The immunoreactive protein bands were visualized with a chemiluminesence solution enhanced with the LimiFlash™ Ultima Chemiluminescent substrate, HRP System (Energenesis Biomedical Co., Ltd.). ImageJ version 1.51 software (National Institutes of Health) was used for densitometry.

HNCCLs were seeded in 96-well plates, with 1.5×10⁴ cells (YD-10B), 2.5×10⁴ cells (SNU-1066) and 2×10⁴ cells (SNU-1041 and RPMI-2650) per well. They were cultured in RPMI-1640 (Welgene, Inc.) supplemented with 0.1% FBS and then treated with 0, 1, 5 or 10 µM concentrations of NE (MilliporeSigma) and 1 µM of propranolol (MilliporeSigma). For blocking, 1 µM propranolol was added to the HNCCLs 1 h before adding 10 µM of NE. The treated HNCCLs were then incubated at 37°C for 24 h. The concentrations of NE and propranolol were selected based on references describing similar protocols (18,21,26,27). Furthermore, cell viability and proliferation were evaluated using the enhanced cell viability assay EZ-CYTOX (DoGen Bio Co., Ltd.) and the Cytoselect™ BrdU cell proliferation enzyme-linked immunosorbent assay kits (cat. no. CBA-251; Cell Biolabs, Inc.). Each experiment was performed thrice independently. The results are expressed as the mean ± standard error of the mean of three replicates.

Among the variables, significantly associated clinical factors were identified using the Mann-Whitney U test, Kruskal-Wallis test and Bonferroni's method. Significant changes in cell viability and proliferation assays were assessed by Kruskal-Wallis along with a post hoc test (Mann-Whitney U test with Bonferroni's method). P<0.05 was considered to indicate a statistically significant difference. All statistical data were analyzed using the SPSS version 22.0 software (IBM, Corp.).

Table I summarizes the demographics of 57 patients with head and neck cancer. All patients displayed β2-AR expression, except for two patients with pharyngeal cancer (55/57; 96.5%). The mean IRS of β2-AR expression was moderately positive (mean ± standard deviation, 6.63±3.30), with no significant differences in relation to sex, T stage, N stage, HPV status, and overall stages. However, it was significantly associated head and neck cancer subsites (P=0.031; Table I). In the post-hoc analysis, oral cavity cancer (P=0.023) had a significantly higher IRS of β2-AR expression than pharyngeal cancer (Table II and Fig. 1).

All HNCCLs showed the mRNA and protein expression of β2-AR. Especially, mRNA expression of β2-AR was high in an oral cavity cancer cell line (Fig. 2A). In western blot analysis, its protein expression was also strong in the same cell line (Fig. 2B and C).

The viability and proliferation of all cancer cells were induced by NE in a dose-dependent manner. These differences were all statistically significant except for in pharyngeal cancer (Figs. 3 and 4). Following post hoc analysis, the cell viability and proliferation in the 10 µM of NE group were significantly higher compared with the 0 µM of NE group. The amplified cell viability and proliferation were significantly inhibited by propranolol treatment in all cancer types (Figs. 3 and 4).