Tocris Bioscience supplied the NE and propranolol (PRO). Western blotting antibodies, including phospho-ERK1/2 (Thr202/Tyr204)) (cat. no. 4370; 1:2,000), ERK1/2 (cat. no. 4695; 1:2,000), phospho-CREB (Ser133) (cat. no. 9198; 1:2,000), CREB (cat. no. 9197; 1:2,000), β-actin (cat. no. 4970; 1:5,000), and anti-rabbit IgG HRP-linked antibody (cat. no. 7074, goat anti-rabbit, 1:5,000) were purchased from Cell Signaling Technology, Inc. ADRB1 (cat. no. 28323-1-AP; 1:2,000) and ADRB2 (cat. no. 29864-1-AP; 1:2,000) antibodies were purchased from ProteinTech Group, Inc. All cell culture reagents were purchased from Gibco (Thermo Fisher Scientific, Inc.). SYBR^® Premix Ex Taq™ II was purchased from Takara Bio, Inc. MTT was purchased from MilliporeSigma.

HepG2 (hepatoblastoma), Huh7 (hepatoma), and LO2 (normal liver) cells were purchased from iCell Bioscience Inc. Both cell lines were cultured in DMEM supplemented with 4.5 g/l glucose, 10% FBS, 50 µg/ml streptomycin, and 50 IU/ml penicillin. The cells were regularly tested for mycoplasma and authenticated using Short Tandem Repeat (STR) analysis to confirm their identity. The STR profiles of both cell lines matched the reference profiles provided by the cell bank. Additionally, their growth characteristics and morphology were consistent with the reported characteristics of HepG2 and LO2 cells. Cells were maintained at 37°C in an incubator supplied with 5% CO₂ air. Small interfering (si)RNAs were used to knock down CREB expression in HepG2 cells. The cells were transfected for 48 h using Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.) with siRNAs against CREB (Santa Cruz Biotechnology, Inc.) according to the manufacturer's instructions.

Cells were added to 24-well plates with culture media and incubated at 37°C for 24 h. After the cells had adhered, the cells were serum-starved overnight and treated for 48 h with various treatments. The culture media was then replaced with supplemented medium containing 50 µl MTT reagent (5 mg/ml) in each well. After a further 3 h of incubation at 37°C, the supernatant was removed, and 500 µl dimethyl sulfoxide was added to each well and shaken for 10 min to dissolve the precipitate. The optical density (OD) at 490 nm was then measured. The cell viability is represented as a percentage of the control (100%).

Cells were serum-starved overnight at 37°C prior to treatment. After treatment, the cells were lysed with RIPA buffer on ice to extract total protein. A total of 10 µg protein/lane was loaded and resolved using 10% SDS-PAGE before transfer to a PVDF membrane and blocked for 2 h at room temperature in 5% fat-free milk. Subsequently, the membrane was treated with one of the primary antibodies against ADRB1, ADRB2, phospho-ERK1/2, ERK1/2, phospho-CREB, CREB, or β-actin overnight at 4°C, followed by incubation with the HRP-conjugated anti-rabbit IgG secondary antibody for 2 h at room temperature. Signals were visualized using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.). Densitometry analysis was performed using ImageJ (version 1.47t; National Institutes of Health).

Wound healing assays were used to assess cell migration. Briefly, HepG2 cells were seeded into a 6-well plate with complete growth medium containing 10% FBS and incubated until they reached 100% confluence. The cells were then cultured in serum starved DMEM (2% FBS) for 12 h. Subsequently, a sterile yellow pipette tip was used to generate a wound, after which, the cells were washed with PBS to remove any floating cells and debris. After adding NE, the cells were cultured in DMEM containing 2% FBS at 37°C. The width of the wound was measured at 0, 12, and 24 h post-scratch, and images of randomly selected fields from each group were captured using a phase-contrast microscope (Olympus Corporation).

Using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was isolated from HepG2 and LO2 cells. The RNA purity and concentration were measured according to the ratio of absorbance at 260 and 280 nm, using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). The RNA was then reverse-transcribed into cDNA using the PrimeScript^® RT reagent (Takara Bio, Inc.) according to the manufacturer's protocol. The thermocycling protocol was as follows: Amplification step, 94°C for 5 min; followed by 35 cycles of denaturation for 1 min at 94°C; 1 min of annealing at 55°C; elongation at 72°C for 1 min; with a final extension step at 72°C for 1 min. The sequences of the primers are shown in Table SI. Standard electrophoresis was performed on a 1.2% agarose gel at 100 V for 40 min. The bands in the gels were imaged using an UV light transilluminator (ChemiDoc XRS+ system; Bio-Rad Laboratories, Inc.). β-actin was used as the housekeeping gene.

The total RNA and cDNA were obtained as above and qPCR was performed on a ViiA7 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). A total of 20 µl qPCR reaction system contained 6 µl nuclease-free water, 10 µl SYBR Premix Ex Taq II (2X), 0.4 µl ROX Reference Dye II, 2 µl cDNA, 0.8 µl forward primer (10 µM) and 0.8 µl reverse primer (10 µM). The thermocycling conditions were: 95°C for 30 sec; followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 60°C for 34 sec, elongation at 95°C for 15 sec, and extension at 60°C for 1 min. The 2^−ΔΔCq method was used to analyze the expression levels of β₁-AR and β₂-AR (24); β-actin was used as the housekeeping gene. Each sample was assessed in triplicate.

The HepG2 cells were seeded into 6-well plates (1×10⁶ cells) in supplemented media. Following adherence of cells, NE was added, and the cells were cultured in DMEM without FBS for 96 h. Random field images were selected and analyzed for morphological changes using ImageJ software (version 1.47t; National Institutes of Health).

Data are presented as the mean ± SEM of at least three separate experiments and were analyzed using GraphPad Prism version 6.0 (GraphPad Software, Inc.). Data were compared using a Student's t-test (2 groups) or a one/two-way ANOVA with Bonferroni's Multiple Comparison Corrections (>2 groups). P<0.05 was considered to indicate a statistically significant difference.

To investigate the expression of β₁-AR and β₂-AR in HepG2 and LO2 cells and compare the differences in the expression of these receptors between the cell lines, RT-PCR and RT-qPCR assay was used. The results indicated that β₁-AR and β₂-AR expression was detectable in HepG2 and LO2 cells (Fig. 1A). Western blotting was further performed to confirm the results (Fig. 1B and C). Notably, the expression of β₂-AR was higher in HepG2 cells than that in the LO2 cells, and the expression of β₂-AR was evidently higher than that of β₁-AR in HepG2. These findings showed that β-AR, particularly β₂-AR, may play an important role in the tumorigenesis and development of liver cancer.

To further investigate the activity of the β-ARs in HepG2 and LO2 cells, cells were treated with the β-AR agonist NE. Cells were treated with different doses of NE for 48 h, and cell viability was assessed using an MTT assay. The results demonstrated that NE significantly promoted the proliferation of HepG2 cells in a dose-dependent manner, with 10 µM concentration showing the most pronounced effect. However, NE did not notably affect the proliferation of LO2 cells (Fig. 2A and B). To confirm the effect of NE on liver cancer, Huh7 liver cancer cells were treated with different doses of NE, and a significant increase in the proliferation of these cells was observed (Fig. S1). Next, the effect of NE on cell viability after treatment of HepG2 cells with NE for 24, 48, or 72 h was assessed. NE increased HepG2 cell growth in a dose and time-dependent manner (Fig. 2C). Additionally, HepG2 cells were co-treated with the non-selective β-AR blocker PRO and NE for 48 h. The results indicated that PRO blocked NE-induced cell growth, suggesting that the proliferative effect of NE may be mediated through the activation of β-AR (Fig. 2D).

As it was demonstrated that β-AR activation may have exerted pro-proliferative effects of NE in HepG2, the specific molecular mechanisms were next explored. It has been reported that the MAPK/ERK1/2 pathway is crucial in regulating key processes, such as cell proliferation, survival, and metastatic progression (25). To determine whether ERK1/2 and CREB were implicated in β-AR activation, HepG2 cells were treated with NE, and then, the phosphorylation levels of ERK1/2 and CREB were measured by western blot analysis. NE induced a brief and transitory increase in ERK1/2 phosphorylation but had no effect on ERK1/2 expression levels (Fig. 3A and B). The phosphorylation of ERK1/2 peaked at 10 min and then decreased. Similarly, the transcription factor CREB, which affects processes such as cell cycle, apoptosis, and cellular metabolism, was also transiently phosphorylated and peaked within 10 min (Fig. 3C).

To confirm that ERK1/2 and CREB phosphorylation was a result of β-AR activation, HepG2 cells were pre-treated with PRO and then stimulated with NE. PRO inhibited the phosphorylation of ERK1/2 and CREB (Fig. 3D-F). As CREB is a key downstream target of ERK1/2, whether NE-induced CREB phosphorylation was mediated by ERK1/2 was assessed. The MEK1/2 inhibitor U0126 was used to treat HepG2 cells. Inhibiting the MAPK pathway significantly inhibited NE-induced ERK1/2 and CREB phosphorylation, suggesting that NE stimulation of β-AR promoted ERK1/2 phosphorylation, which then activated CREB (Fig. 3G-I).

Considering the potential of NE to significantly increase HepG2 cell proliferation and activate ERK1/2 and CREB phosphorylation, HepG2 cells were pre-treated with U0126 (a selective inhibitor of ERK) and then treated with NE to further demonstrate whether NE-mediated ERK1/2 and CREB activation were involved in cell proliferation. U0126 inhibited the proliferative effects of NE (Fig. 4A). Additionally, knocking down CREB expression using specific siRNAs significantly reduced HepG2 cell growth compared with the control siRNA-transfected cells (Fig. 4B and C). Together, this suggested that ERK1/2 was involved in NE-mediated proliferation in HepG2 cells via CREB phosphorylation.

Previous studies have reported that NE promotes tumor metastasis in colon cancer and other types of cancer (26,27). Therefore, the role of β-ARs in cell migration in HepG2 cells was investigated. The wound healing assays showed that NE had no significant effect on HepG2 cell migration compared with the control group (Fig. 5A).

It has also been shown that β-AR is involved in the EMT process in oral squamous cell carcinoma and glioma cells (28,29). To determine whether β-ARs were involved in the EMT process of HepG2 cells, the morphological changes of the cell nuclei in NE-treated HepG2 cells were observed under a phase-contrast microscope. The results showed that NE treatment did not induce significant morphological changes in HepG2 cells (Fig. 5B). Subsequently, cellular RNA was extracted, and RT-qPCR was used to evaluate the expression of the EMT markers, E-cadherin, and Vimentin. The results demonstrated that NE had no effect on the expression of E-cadherin and Vimentin in HepG2 cells (Fig. 5C).

There is growing evidence that NE treatment can regulate the expression of genes related to the cell cycle and proliferation, thereby regulating the occurrence and development of tumors (30). To determine the effect of β-AR on gene expression in HepG2 cells, HepG2 cells were treated with NE, and the expression of cyclinE2, Ki67, P53, P27, HIF-1α, COX-2, and VEGF in HepG2 cells was detected. The results showed that β-AR activation significantly increased the expression of these genes (Fig. 6).