DMEM and RPMI-1640 medium, as well as fetal bovine serum (FBS), were purchased from Gibco (Thermo Fisher Scientific, Inc.). PrimeScript™ RT reagent kit (cat. no. RR037A) and One Step TB Green^® PrimeScript™ RT-PCR kit (cat. no. RR066A) were obtained from Takara Bio, Inc. Transwell chambers and Matrigel were purchased from BD Biosciences. Rabbit anti-human CXCR4 (cat. no. ab124824), rabbit anti-human total (t-)P65 (cat. no. ab16502), rabbit anti-human phosphorylated (p-)P65 (cat. no. ab86299) and rabbit anti-human β-actin (cat. no. ab179467) primary antibodies, as well as horseradish-peroxidase (HRP)-conjugated goat anti-rabbit (cat. no. ab6721) and HRP-conjugated goat anti-mouse (cat. no. ab6789) secondary antibodies, recombinant human SDF1 protein (cat. no. ab9798) and goat anti-rabbit IgG H&L (Alexa Fluor^® 555; cat. no. ab150078) were purchased from Abcam. The primary antibodies targeting IκBα (cat. no. 9242), p-IκBα (Ser32/36; cat. no. 9246) and histone H3 (cat. no. 9715) were obtained from Cell Signaling Technology, Inc. PCR primers were synthesized by Shanghai Sangon Biotech Co., Ltd. TRIzol^® reagent (cat. no. 15596026) and NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (cat. no. 78833) were purchased from Thermo Fisher Scientific, Inc. The NF-κB activation inhibitor JSH-23 (cat. no. 481408-M) was obtained from Sigma-Aldrich (Merck KGaA).

The HepG2 and Huh7 human liver cancer cell lines were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences and maintained within our laboratory. HepG2 cells were cultured in DMEM containing 10% FBS and 1% streptomycin. Huh7 cells were cultured in RPMI-1640 containing 10% FBS and 1% streptomycin. Both cell lines were maintained in a humidified incubator at 37°C with 5% CO₂, and passaged using 0.25% trypsin EDTA at a confluence of 80-90%. Tumor cells were treated with either cordycepin alone or in combination with the NF-κB pathway inhibitor, JSH-23. Untreated cells were used as the control.

Cordycepin powder (Santa Cruz Biotechnology, Inc.) was dissolved in DMSO to prepare a 10 mM stock solution, which was then diluted with medium to 1, 5 and 10 µM working solutions. JSH-23 was dissolved in DMSO to produce a 1 mM stock solution, and diluted to a working concentration of 1 µM using the appropriate medium for each cell type.

HepG2 and Huh7 cells were seeded into a 6-well plate at a density of 8×10⁵ cells per well. At ~50% confluency, 1, 5 or 10 µM cordycepin and/or 1 µM JSH-23 was added and the cells were further incubated for 72 h. The cells were then digested using trypsin, and resuspended in serum-free medium; 1×10⁴ cells per well were added to the upper chamber of the Transwell insert, and 500 µl medium (with 10% FBS) was added to the lower chamber. Following a further 8 h incubation period, the upper chamber was removed and the unmigrated cells were wiped away with a cotton swab. Finally, the cells on the lower chamber were stained with 0.1% crystal violet at 37°C for 15 min, washed, air-dried and counted under an inverted light microscope prior to being photographed. All experiments were repeated three times.

Matrigel working solution (100 µl; 500 ng/ml) prepared using serum-free medium was used to pre-coat the polycarbonate membrane of the upper chamber of a Transwell insert. After a 2 h incubation at 37°C, the residual basal medium was removed. The subsequent steps were performed similar to the aforementioned migration assay. Briefly, HepG2 and Huh7 cells (8×10⁵ cells per well) were seeded into a 6-well plate and treated with cordycepin or JSH-23 for 72 h. The cells were subsequently added to the upper chamber at a density of 1×10⁴ cells per well, and 500 µl medium (10% FBS) was added to the lower chamber. The cells were incubated for 24 h, stained and photographed as aforementioned.

As described in the migration assay, cells were seeded into the upper chamber of a Transwell insert at a density of 1×10⁴ cells per well, and incubated for 12 h; 500 µl medium (serum-free) containing recombinant human SDF1 (500 ng/ml) was added to the lower chamber. After staining with 0.1% crystal violet, the cells were visualized under an inverted light microscope as aforementioned.

Total RNA was extracted from HepG2 and Huh7 cells treated with cordycepin and/or JSH-23 using TRIzol^® reagent, according to the manufacturer's instructions. The RNA concentration and purity were measured using an ultraviolet spectrophotometer, and A260/A280 in the range of 1.8-2 was regarded as acceptable. Total RNA (1 µg) was reverse transcribed into cDNA using the PrimeScriptTM RT reagent according to the manufacturer's instructions. The reverse transcription conditions were 37°C for 15 min, followed by 85°C for 5 sec, and storage at 4°C. Subsequently, qPCR was conducted using a Bio-Rad qPCR instrument (Bio-Rad Laboratories, Inc.) with β-actin as the internal reference. The reaction procedure was as follows: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 15 sec, and annealing at 60°C for 15 sec (repeated for 40 cycles). The relative expression levels of the target gene were calculated using the 2^−ΔΔCq method (35). Each sample was run in triplicate and all experiments were repeated three independent times. The primer sequences were as follows: CXCR4 forward, 5′-CGGAATTCCAGCAGGTAGCAAAGTGACG-3′ and reverse, 5′-GACGCCAACATAGACCACCT-3′; and β-actin forward, 5′-CCTCGCCTTTGCCGATCC-3′ and reverse, 5′-GGATCTTCATGAGGTAGTCAGTC-3′.

HepG2 and Huh7 cells treated with cordycepin or JSH-23 were harvested for total protein extraction using cell lysis buffer for western and IP (Beyotime Institute of Biotechnology), and the protein concentration was determined using the bicinchoninic acid assay method. The protein samples (30 µg per lane) were separated by 10% SDS-PAGE, transferred onto a PVDF membrane, and blocked with 5% skim milk at room temperature for 2 h. The membranes were incubated with the primary antibodies overnight at 4°C (β-actin dilution 1:4,000; all other primary antibodies diluted 1:1,000). The membrane was then incubated with the secondary antibody at room temperature for 2-4 h (1:5,000). After washing with TBS/0.1% Tween-20, the bands were visualized using enhanced chemiluminescence solution and images were captured using a gel imager (Bio-Rad Laboratories, Inc.). The gray values were analyzed using Bio-Rad Image Lab Software 5.1 (Bio-Rad Laboratories, Inc.).

HepG2 and Huh7 cells (5-10×10⁶) treated with cordycepin and/or JSH-23 were collected and washed with cold PBS via centrifugation at 8,000 × g for 10 min (4°C). After discarding the supernatant, the cell pellets were resuspended in pre-cooled Cytoplasmic Extraction Reagent (CER) I, followed by a 10 min incubation on ice. The cells were then vortexed for 5 sec with pre-cooled CER II reagent, and incubated on ice for 1 min. Following centrifugation at 15,000 × g for 10 min, the supernatants (cytoplasmic fraction) were immediately transferred to a pre-chilled EP tube. The pellets were resuspended in pre-cooled Nuclear Extraction Reagent for 40 min on ice, with vortexing at 15 min intervals. Finally, the solution was centrifuged for 10 min at 15,000 × g (4°C), and the supernatants (nuclear fraction) were transferred to a clean pre-cooled EP tube. The nuclear extracts were subsequently analyzed by western blotting.

HepG2 and Huh7 cells were cultured on sterile slides and treated with cordycepin and/or JSH-23. The slides were washed with PBS three times for 5 min each, and then fixed with 4% paraformaldehyde for 30 min at room temperature. The slides were washed for a further three times for 5 min each with PBS, followed by permeabilization with PBS/0.25% Triton for 10 min. The slides were then washed once more (PBS, three times for 5 min each) and blocked with 5% bovine serum albumin (BSA) solution for 1 h. Each sample was incubated with rabbit anti-human P65 primary antibody (1:300, diluted in 5% BSA) at 4°C for 12-24 h, washed with PBS three times, and then incubated with goat anti-rabbit Alexa Fluor^® 555 secondary antibody (1:300, diluted in 5% BSA) at room temperature for 2-3 h. Following further washing with PBS, the slides were stained with DAPI solution (1:10,000) for 5-10 min. Following a final round of washing with PBS, 50% glycerol were added onto each coverslip and the cells were observed under a fluorescence microscope.

Data analysis was conducted using SPSS 21.0 (IBM Corp.) and GraphPad Prism 6.0 (GraphPad Software, Inc.). Mean comparisons among multiple groups were performed using one-way analysis of variance with Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

As described previously (36), relatively high concentrations of cordycepin induce apoptosis and inhibit proliferation of HepG2 cells. To determine an appropriate concentration that has no significant effect on the proliferation of HepG2 and Huh7 cells, a dose-curve experiment was performed in the present study. When the concentration of cordycepin was 1, 5 and 10 µM, no significant difference was observed in the proliferation of HepG2 and Huh7 cells by CCK-8 assay, compared with the untreated cells (Fig. 1A). Additionally, as presented in Fig. 2, the migratory and invasive abilities of HepG2 (Fig. 2A-D) and Huh7 cells (Fig. 2E-H) treated with these concentrations of cordycepin (1, 5 and 10 µM) were significantly inhibited (P<0.05) in a dose-dependent manner (with the exception of Huh7 cells treated with 1 µM cordycepin; P>0.05), compared with the untreated controls. Finally, compared with untreated cells, a significant dose-dependent downregulation of CXCR4 expression was observed by RT-qPCR and western blot assays (P<0.05; Fig. 2I-N). Therefore, three different concentrations of cordycepin (1, 5 and 10 µM) were selected for subsequent experiments, which suppressed the cell migratory and invasive capabilities, but had no significant effect on cell proliferation.

Human liver cancer cells were treated with 1, 5 and 10 µM cordycepin. The expression and activation of NF-κB signaling pathway-related proteins, including t-IκBα, p-IκBα, t-P65 and p-P65, were evaluated by western blotting. As demonstrated in Fig. 3, different concentrations of cordycepin significantly downregulated the activation of IκBα and P65, and the expression levels of p-IκBα and p-P65 (P<0.05). No significant differences were observed in the expression levels of t-IκBα and t-P65.

To exclude the possibility of an effect of JSH-23 on the viability of HepG2 and Huh7 cells and to select a suitable drug concentration (37), a CCK-8 assay was performed. The results indicated that concentrations up to 1 µM of JSH-23 did not significantly alter cell proliferation compared with the untreated cells (Fig. 1B). As presented in Fig. 4, a significant increase in t-P65 was observed in the cytoplasm of HepG2 (Fig. 4A and B) and Huh7 (Fig. 4C and D) cells following treatment with 10 µM cordycepin (P<0.05), as well as a corresponding reduction in the nuclear fraction (P<0.05), compared with the untreated-control groups. Immunofluorescence analysis indicated that the co-administration of 1 µM JSH-23 and 10 µM cordycepin markedly inhibited the nuclear translocation of P65 in HepG2 (Fig. 4E) and Huh7 cells (Fig. 4F).

The results of the present study preliminarily suggested that cordycepin inhibited the phosphorylation of IκB, as well as the activation and nuclear localization of P65, altering the expression of its downstream gene, CXCR4. As demonstrated in Fig. 5, treatment with 10 µM cordycepin and/or 1 µM JSH-23 suppressed the migration (Fig. 5A, B, E and F) and invasion (Fig. 5C, D, G and H) capacities of liver cancer cells compared with untreated controls (P<0.05). Furthermore, the combination treatment revealed a synergistic inhibitory effect (P<0.05; Fig. 5) compared with cordycepin treatment alone.

In order to understand the underlying synergistic mechanism between cordycepin and JSH-23, the expression of NF-κB signaling pathway-associated proteins (t-IκBα, p-IκBα, t-P65 and p-P65) was detected using RT-qPCR and western blotting. As shown in Fig. 6, treatment with cordycepin (10 µM) alone reduced IκBα and P65 phosphorylation in HepG2 and Huh7 human liver cancer cells (P<0.05). The co-treatment with cordycepin (10 µM) and JSH-23 (1 µM) significantly inhibited the expression of CXCR4 (P<0.05; Fig. 6), compared with cordycepin treatment alone. No significant effect on the expression levels of t-IκBα and t-P65 was observed by any of the indicated treatments (Fig. 6).

SDF1, a specific ligand for CXCR4, was used to detect the chemotactic ability of human liver cancer cells treated with 10 µM cordycepin and/or 1 µM JSH-23 via Transwell assays. Fig. 7 illustrates that cordycepin and JSH-23 monotherapy significantly reduced the chemotactic sensitivity of the cells to SDF1 (P<0.05), and that combination treatment further enhanced this effect compared with the control cells (P<0.05).