The BxPC-3 human pancreatic cancer cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The human hepatoblastoma HepG2 cell line (13) was generously provided by Dr Chang Liu (Medical College, Xi'an Jiaotong University). The SKOV-3 ovarian carcinoma cell line was kindly provided by the First Affiliated Hospital Obstetrics and Gynecology Laboratory at Xi'an Jiaotong University. BxPC-3 and HepG2 cells were cultured in DMEM, and SKOV-3 cells were cultured in RPMI-1640 medium. The medium was supplemented with 10% fetal bovine serum (FBS), and cells were maintained under normal culture conditions of 5% CO₂ at 37°C. Res, norepinephrine (NE), HIF-1α antibody (diluted 1:1,000, ab51608), ADRB-2 antibody (diluted 1:1,000, ab61778) and MTT were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Proliferating cell nuclear antigen (PCNA) and Ki-67 antibodies (both diluted 1:200, sc-56 and sc-15402) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

BxPC-3, HepG2 and SKOV-3 cancer cells were plated in 96-well plates at a density of 5×10³ cells/well and treated with 10 µM norepinephrine (NE). After 24 h, the NE-containing medium was removed and various concentrations (0, 25, 50, 75 and 100 µM) of Res were added to the cells. Cell viability was assessed by the MTT assay at the indicated time points (24, 36 and 48 h). A multi-well microplate reader (BIO-TEC, Inc., Richmond, VA, USA) was used to measure the absorbance at 490 nm.

Cells (1,000 cells/well) were seeded in triplicate and allowed to adhere overnight, and they were then incubated with NE for 48 h. Subsequently, the cells were treated with Res without NE-containing medium for 24 h followed by incubation with media lacking Res for 7–10 days. The cells were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution, and the colonies (>50 cells) were counted.

Apoptosis was determined by flow cytometry with an Annexin V-FITC/PI apoptosis detection kit (Beyotime Institute of Biotechnology, Shanghai, China). Briefly, cells were seeded at density of 1×10⁵ cells/well in 6-well plates and cultured to the indicated density followed by treatment with 10 µM NE for 24 h. Cells were treated with fresh medium containing various concentrations (0, 25 and 50 µM) of Res for an additional 24 h. Subsequently, the cells were trypsinized, washed with phosphate-buffered saline (PBS) and stained with Annexin V and PI. Apoptosis was evaluated using flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA).

For analysis of the HIF-1α, ADRB-2, proliferating cell nuclear antigen (PNCA) and Ki-67 protein expression, cell lysates were prepared from BxPC-3, HepG2 and SKOV-3 cells, which had been treated with medium containing 10 µM NE for 24 h followed by treatment with fresh medium containing various concentrations (0, 25 and 50 µM) of Res. Total proteins were extracted using RIPA lysis buffer (Sigma Aldrich; Merck KGaA), separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto PVDF membranes. After blocking with 5% non-fat milk in Tris-buffered saline Tween-20 (TBST) for 1 h, the membranes were incubated with primary antibody overnight at 4°C. The primary antibody was removed, and the membranes were then incubated with 1:2,000 horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Protein expression was visualized with enhanced chemiluminescence, and images were captured using the ChemiDoc XRS imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Quantity One image software was used for the densitometry analysis of each band. β-actin was used as an internal loading control.

Total cell RNA was extracted using TRIzol reagent following the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). cDNA synthesis was performed using a PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed with an iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) using a SYBR-Green PCR kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. The amplification consisted of predenaturation at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec and extension at 72°C for 30 sec for 32 cycles. After the last cycle, a final extension was performed at 72°C for 7 min. The primer sequences for ADRB-2 were as follows: Forward 5′-GGGTCTTTCAGGAGGCCAAA-3′ and reverse 5′-ATGCCTAACGTCTTGAGGGC-3′; the primer sequences for HIF-1α were as follows: Forward 5′-CTTGGCAACCTTGGATTGGATG-3′ and reverse 5′-AATCTCCGTCCCTCAACCTCT-3′. The ΔΔCq method was used to calculate the relative expression of the sample genes with β-actin as the normalized reference gene.

Cells were plated in chamber slides, incubated for 24 h, and then treated as indicated. The cells were fixed using 4% paraformaldehyde for 15 min and permeabilized using PBS containing 0.5% Triton X-100 for 20 min at room temperature. The cells were then incubated with the primary antibody overnight at 4°C, followed by incubation with fluorescein-conjugated secondary antibody at room temperature for 1 h. The cells were then visualized using a fluorescence microscope.

Each experiment was performed at least three times. The results are expressed as the mean ± standard deviation of the number of experiments. One-way analysis of variance was used to evaluate the statistical significance between groups. P-value <0.05 was considered to indicate statistically significant differences. All statistical analyses were performed using SPSS software, version 15.0 (SPSS, Inc., Chicago, IL, USA).

To imitate a chronic stress condition in vitro, three cell lines, namely BxPC-3, HepG2 and SKOV-3, were treated with NE. After 24 h, western blotting was used to detect ADRB-2 expression. The results demonstrated that ADRB-2 expression was significantly increased after treatment, indicating that NE may be used for in vitro experiments to simulate chronic stress (Fig. 1).

First, the effects of Res on the viability of chronically stressed cancer cells were examined. Res inhibited chronically stressed cancer cells in a dose- and time-dependent manner (Fig. 2A). Colony formation ability was detected after the addition of Res to chronically stressed cancer cells. Compared with untreated cells, treatment with 25 µM Res markedly decreased the number of colonies, and there was almost no colony formation when the concentration of Res was increased to 50 µM (P<0.05) (Fig. 2B). PCNA is located in the nucleus and plays an important role in cell proliferation. Ki-67 protein expression is used as a marker to indicate cell proliferation. To test whether Res inhibits the proliferation of chronically stressed cancer cells, PCNA and Ki-67 protein expression was measured in the three cell lines. Western blot analysis revealed that Res (0, 25 and 50 µM) suppressed PCNA and Ki-67 expression in chronically stressed cancer cells in a dose-dependent manner (Fig. 2C).

To determine whether Res induces cancer cell apoptosis under chronic stress conditions, flow cytometric analyses were conducted after the cancer cells (BxPC-3, HepG2 and SKOV-3) were treated with Res (25 and 50 µM) for 48 h in the presence of NE. The apoptosis rates of the cancer cells were calculated by summing up the quadrants of the early (lower right) and late (upper right) apoptosis of the FITC graphs. Res increased apoptosis of the three chronically stressed cancer cell types compared with untreated cells (Fig. 3, P<0.05).

Previous studies provided evidence that ADRB-2 plays an important role in chronic stress-induced tumor growth (4). Based on our previous findings, we hypothesized that the inhibition of chronically stressed cancer cells by Res may be mediated by the ADRB-2/HIF-1α axis. To verify the effect of Res on the ADRB-2/HIF-1α axis, RT-qRCR, western blotting and immunofluorescence assays were used to measure ADRB-2 and HIF-1α expression after treatment of chronically stressed BxPC-3, HepG2 and SKOV-3 cancer cells with 0, 25 and 50 µM Res. RT-qRCR demonstrated that Res treatment markedly decreased ADRB-2 and HIF-1α expression at the mRNA level (P<0.05) (Fig. 4A). The protein expression levels of ADRB-2 and HIF-1α were confirmed by western blotting (Fig. 4B). Consistently, the immunofluorescence results revealed that the ADRB-2 and HIF-1α protein levels were markedly decreased following Res treatment (Fig. 4C). Taken together, these findings confirmed that Res inhibits chronically stressed cancer cell proliferation via inhibiting ADRB-2 and HIF-1α expression.