The three-dimensional structures of RSK2 and ERKs were obtained from the PDB databank (http://www.rcsb.org/) [RSK2 N-terminal kinase domain, PDB ID 3UBD (21); RSK2 C-terminal kinase domain, PDB ID 4D9U (22); ERK1 kinase domain, PDB ID 4QTB; and ERK2 kinase domain, PDB ID 4QTA (23)]. Prior to virtual screening, the raw PDB structures were converted into an all-atom, fully prepared receptor model structure with the Protein Preparation Wizard module in Schrödinger (24). Subsequently, the docking receptor grids were created by Glide's Receptor Grid Generation (25). The grid boxes and centers were set to default with the co-crystal ligands in their ATP binding sites.

The structures of >500 traditional Chinese medicine compounds, which were downloaded from the PubChem Compound database (http://www.ncbi.nlm.nih.gov/pccompound/), were downloaded and a small compound database referred to as the ShiLan database was built using the LigPrep module (26). These compounds were available from TianJin ShiLan Technology Company (Tianjin, China). Glide was used to run the extra precision (XP)-docking screening of this ShiLan database targeted to two protein receptor structures (3UBD and 4D9U) (27,28). The screening processes outputted two rank lists of nearly 50 compounds each. The compound IBC (CID5281255) appeared in each list, and was manually selected for further experimental tests.

Flexible ligand-protein docking was performed using the Induced Fit Docking (IFD) Module (29) in Schrödinger to assess the possible binding modes between ERKs/RSK2 and IBC. The induced fit docking can capture the possible conformational changes in receptor active site upon ligand binding. During IFD, the grid box and center for each receptor structure were set to default with the co-crystal ligand in its active binding site. Docking of IBC using its LigPrep's minimized structure into each receptor structure was also performed with Glide in XP mode (28). The binding pose with the lowest docking score was considered as the correct binding structure. The docking structures for each of the ERKs/RSK2-IBC complexes were generated using Maestra (30) in Schrödinger.

IBC (98% purity) was purchased from Tianjin ShiLan Technology Company, and then it was dissolved in dimethyl sulfoxide (DMSO; 100 mM stock solution). CNBr-activated Sepharose 4B and epidermal growth factor (EGF) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The primary antibodies were purchased from the following companies: Mouse monoclonal antibodies against p53 (cat. no. 2524; 1:1,000) and caspase-9 (cat. no. 9508; 1:1,000), rabbit monoclonal antibodies against cleaved caspase-9 (cat. no. 7237; 1:1,000), caspase-7 (cat. no. 12827; 1:1,000), cleaved caspase-7 (cat. no. 8438; 1:1,000), caspase-3 (cat. no. 9665; 1:1,000), cleaved caspase-3 (cat. no. 9664; 1:1,000), poly ADP-ribose polymerase (PARP; cat. no. 9542; 1:1,000), ERK1/2 (cat. no. 5695; 1:2,000), phospho-ERK1/2 (Thr202/Tyr204; cat. no. 4370; 1:1,000), phospho-RSK2 (Ser227; cat. no. 3556; 1:1,000) and Flag tag (cat. no. 14793; 1:1,000), and rabbit polyclonal antibodies against RSK2 (cat. no. 9340; 1:1,000), stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK; cat. no. 9525; 1:1,000), histone H3 (cat. no. 9715; 1:1,000) and phospho-histone H3 (Ser10) (cat. no. 9701; 1:1,000) purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); mouse monoclonal antibodies against mouse double minute 2 homolog (MDM2; cat. no. sc-13161; 1:500), RSK2 (cat. no. sc-9968; 10 µl) and β-actin (cat. no. sc-8432; 1:1,000) purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); rabbit polyclonal antibodies against cAMP response element-binding protein (CREB; cat. no. ab31387; 1:1,000) and activating transcription factor 1 (ATF1; cat. no. ab225880; 1:1,000), and rabbit monoclonal antibodies against phospho-CREB (Ser133; cat. no. ab32096; 1:1,000) and phospho-ATF1 (Ser63; cat. no. ab76085; 1:1,000) purchased from Abcam (Cambridge, MA, USA); mouse monoclonal antibodies against B-cell lymphoma 2 (Bcl-2; cat. no. 610538; 1:1,000) and Bcl-2 associated X protein (Bax; cat. no. 610982; 1:1,000) purchased from BD Transduction Laboratory (BD Biosciences; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Dylight 680-conjugated anti-mouse IgG (cat. no. 610-144-002; 1:10,000) and Dylight 800-conjugated anti-rabbit IgG (cat. no. 611-145-002; 1:10,000) secondary antibodies were purchased from Rockland Immunochemicals, Inc. (Limerick, PA, USA).

The human liver cancer cell lines HepG2 and Hep3B and the normal immortalized liver cell line L02 were obtained from American Type Culture Collection (Manassas, VA, USA). HepG2 and Hep3B cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 100 mg/ml streptomycin, 100 IU/ml penicillin at 37°C in an incubator containing 5% CO₂, and L02 cells were cultured in DMEM with 20% FBS. Cells freshly revived from cryopreservation were maintained and cultured for ≤8 weeks, as aforementioned. Activating protein-1 (AP-1) luciferase reporter vector was provided by Dr ArndKieser (Helmholtz Zentrum München, Munich, Germany). The pCMV3-C-Flag-RSK2 (pCMV3-RSK2) vector and pCMV3-C-Flag (pCMV3) control vector were obtained from Sino Biological Inc. (Beijing, China). To construct the short hairpin RNA (shRNA) vector targeting RSK2 (shRSK2) and the scramble shRNA control vector (shCtrl), the mU6pro vector was digested with XbaI and BbsI. The annealed synthetic primers (shRSK2, sense, 5′-TTTGAAGGCAGATCCTTCCCAGTTTCAAGAGAACTGGGAAGGATCTGCCTTTTTTT-3′, and antisense, 5′-CTAGAAAAAAAGGCAGATCCTTCCCAGTTCTCTTGAAACTGGGAAGGATCTGCCTT-3′; and shCtrl, sense, 5′-TTTGACTACCGTTGTTATAGGTGTTCAAGAGACACCTATAACAACGGTAGTTTTTT-3′, and antisense, 5′-CTAGAAAAAAACTACCGTTGTTATAGGTGTCTCTTGAACACCTATAACAACGGTAGT-3′) were then introduced into the mU6pro vector (31). The recombinant plasmids were confirmed by DNA sequencing. L02 cells (5×105 cells/well) or HepG2 cells (4×105 cells/well) were seeded in 6-well plates and cultured to 60–70% confluence. Then, pCMV3-RSK2 (3 µg), shRSK2 (3 µg) and their corresponding controls were transiently transfected into cells with jetPEI™ DNA transfection reagent (Polyplus-Transfection SA, Illkirch, France), according to the manufacturer's protocols. After being transfected for 24 h, cells were used for subsequent experimentation.

HepG2, Hep3B or L02 cells were seeded into 96-well plates at a density of 3×10³ cells/well, and then incubated with 5, 10, 20 or 40 µM IBC for 24 or 48 h at 37°C. Cell viability was determined with a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Laboratories, Inc., Kumamoto, Japan) assay. A total of 10 µl CCK-8 solution was added to each well and cells were incubated for 2 h at 37°C. The absorbance was measured at a wavelength of 450 nm using a Synergy 2 Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). The half-maximal inhibitory concentration (IC₅₀) of IBC was calculated by Probit analysis using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA).

Cell proliferation was assessed using a Cell-Light EdU DNA Cell Proliferation kit (Guangzhou RiboBio Co., Ltd., Guangzhou, China). HepG2 or Hep3B cells were seeded into 96-well plates at a density of 3×10³ cells/well, and then incubated with 10 or 20 µM IBC for 48 h at 37°C. Following treatment with IBC, cells were exposed to 50 µM EdU for 2 h at 37°C, and then fixed with 4% formaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Subsequently, cells were incubated with 100 µl 1X Apollo^® reaction cocktail for 30 min at room temperature, followed by Hoechst 33342 (5 µg/ml) for 30 min at room temperature in the dark. The stained cells were imaged under a fluorescence microscope (Olympus Corporation, Tokyo, Japan) at a magnification of ×200, and the EdU positive ratio was calculated as (EdU-labeled cells/Hoechst-stained cells) × 100%.

HepG2 or Hep3B cells were seeded in 6-well plates at a density of 500 cells/well and cultured with DMEM complete medium containing 5 or 10 µM IBC for 2 weeks at 37°C. The cell colonies were fixed with methanol for 15 min at room temperature and then stained with 0.5% crystal violet for 15 min at room temperature. The number of colonies containing ≥50 cells was counted under an inverted optical microscope at a magnification of ×40.

HepG2 or Hep3B cells were starved in serum-free DMEM medium for 24 h at 37°C and treated with 10 or 20 µM IBC for another 48 h at 37°C. Subsequently, cells were harvested with trypsin and fixed with 70% ice-cold ethanol at 4°C overnight. The fixed cells were stained via incubation with 10 µg/ml propidium iodide (PI) and 100 µg/ml RNase for 30 min at room temperature in the dark. For the apoptosis analysis, cells were treated with 10 or 20 µM IBC for 48 h at 37°C and stained with Annexin V-fluorescein isothiocyanate and PI (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) for 15 min at room temperature in the dark, according to the manufacturer's instructions. All of the samples were analyzed using a FACS Calibur flow cytometer with Cell Quest software version 3.0 (BD Biosciences; Becton, Dickinson and Company).

HepG2 or Hep3B cells were harvested and total protein was extracted using Radioimmunoprecipitation Assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) with phenylmethane sulfonyl fluoride (PMSF). The isolation of histone protein was performed as described previously (32). Protein concentration was determined using a bicinchoninic acid kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of total protein (50 µg) or histone protein (20 µg) were resolved by 10 or 15% SDS-PAGE, respectively, and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated with blocking buffer (PBS with 5% non-fat milk and 0.1% Tween-20) for 1 h at room temperature, and then probed with specific primary antibodies overnight at 4°C. Subsequently, the membranes were incubated with the species-appropriate infrared-dye-conjugated secondary antibodies for 2 h at 4°C, and then protein bands were visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

CNBr-activated Sepharose 4B beads (0.1 g) were washed five times with 1 mM HCl and collected by centrifugation at 500 × g for 3 min at 4°C. Subsequently, the beads were incubated with 3 mg IBC or DMSO in coupling buffer [0.1 M NaHCO₃ and 0.5 M NaCl (pH 8.3)] with gentle rocking overnight at 4°C. The beads were then washed five times with coupling buffer to remove excess ligand and incubated in blocking buffer [0.1 M Tris-HCl (pH 8.0)] overnight at 4°C. Afterwards, the beads were alternatively washed in three cycles with 0.1 M acetic acid buffer (pH 4.0) containing 0.5 M NaCl and 0.1 M Tris-HCl (pH 8.0) containing 0.5 M NaCl, and then resuspended in 500 µl PBS. IBC-Sepharose 4B beads (or only Sepharose 4B as a control) were then incubated with cellular supernatant fractions of HepG2 or Hep3B cells (500 µg) in reaction buffer [50 mM Tris (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP40 and 0.02 mM PMSF] containing 2 µg/ml BSA and protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany) with gentle rocking overnight at 4°C. Finally, the beads were washed five times with reaction buffer, and the bound proteins were visualized by western blotting according to the aforementioned protocol.

The pCMV3-RSK2 expression vector was transiently introduced into HepG2 cells, and total protein was extracted using IP lysis buffer (Beyotime Institute of Biotechnology). Cell extracts (300 µg) were incubated with 2 µg anti-Flag tag antibody overnight at 4°C, followed by incubation with 20 µl of protein A/G agarose beads (Santa Cruz Biotechnology, Inc.) for 2 h at 4°C. The precipitated beads were washed three times with lysis buffer and twice with 1X kinase buffer (Cell Signaling Technology, Inc.). The kinase reactions were performed in a total of 40 µl 1X kinase buffer supplemented with 200 µM ATP (Cell Signaling Technology, Inc.), 1 µg recombinant human histone H3 (New England BioLabs, Inc., Ipswich, MA, USA), and 10 or 20 µM IBC at 30°C for 30 min. Reactions were terminated with 6X SDS sample buffer (Beyotime Institute of Biotechnology), and then the supernatants were boiled for 5 min at 95°C and resolved by 15% SDS-PAGE. The phosphorylation levels of histone H3 were detected by western blotting according to the aforementioned protocol.

The effect of IBC on the endogenous RSK2 activity was further analyzed. HepG2 cells were starved in serum-free DMEM medium for 24 h at 37°C and treated with 10 or 20 µM IBC for 2 h prior to exposure to EGF (10 ng/ml) for 15 min at 37°C. Cell extracts (500 µg) were used for IP with RSK2 antibody according to the aforementioned protocol, and then an in vitro kinase assay was performed with histone H3 peptide as a substrate.

HepG2 or Hep3B cells were seeded into 24-well plates at a density of 1×10⁵ cells/well, and then were co-transfected with AP-1 luciferase reporter vector (1.0 µg) and pRL-TK Renilla luciferase vector (0.02 µg) (Promega Corporation, Madison, WI, USA) using jetPEI DNA transfection reagent. At 24 h after transfection, cells were serum-starved for another 12 h and pretreated with 10 or 20 µM IBC for 4 h at 37°C, followed by stimulation with EGF (10 ng/ml) for 12 h at 37°C. Cells were lysed with passive lysis buffer (Promega Corporation) for 20 min with gentle shaking, and then the firefly luciferase and Renilla luciferase activities were measured using a Dual-Luciferase assay system (Promega Corporation) in a FB12 Luminometer from Titertek-Berthold (Berthold Detection Systems GmbH, Pforzheim, Germany). The firefly luciferase activity was normalized to Renilla luciferase activity to equalize the transfection efficiency.

All data were analyzed using SPSS 16.0 and GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Data from at least three independent experiments are expressed as the mean ± standard deviation. An unpaired Student's t-test was employed to evaluate the difference between two groups. The differences were determined using one-way analysis of variance with Dunnett's post hoc corrections for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

IBC is a prenylated chalcone of the class flavonoid (Fig. 1A). The predicted docking structures of IBC into the binding pockets of ERKs and RSK2 kinases are depicted in Fig. 1B. Additionally, the ligand-protein interactions of IBC-ERKs/RSK2 complexes in two-dimensional diagrams are further depicted in Fig. S1. IBC docked inside the ATP binding pocket of ERK1 (Fig. 1B-a) and formed five hydrogen-bonds with ERK1, with three involved in the backbone atoms of the residues of Asp123, Met125 and Lys72, while the other two engaged with the side-chain atoms of the residues of Lys71 and Tyr53 in close proximity to the ATP binding pocket (Fig. 1B-b and S1A). Among these five residues, Asp123 and Met125 were located in the hinge loop and Tyr53 was the gate residue in the glycine-rich loop, which also developed π-interactions with IBC between their benzene rings (Fig. 1B-b and S1A). Additionally, the 3-methyl-2-butene tail of the ligand located inside a hydrophobic pocked formed by the residues of Tyr53, Ile73, Ile89 and Ile120, while the other carbon atoms of IBC also created the hydrophobic interaction with the side-chains of the residues Ile48, Tyr53, Met55, Val56, Ala69, Ile101, Leu124, Met125 and Leu173 around the ATP binding site (Fig. S1A). The docking of this compound to ERK2 provided a similar binding pose as that between IBC and ERK1.

However, the docking of IBC to the N-terminal kinase domain (NTKD) of RSK2 provided a binding pose (Fig. 1B-c) similar to the SL0101/afzelin-bound form (21), which demonstrated notable structural rearrangements of the N-lobe, compared with the AMP-PNP-bound form (33) as a typical molecular architecture of AGC kinases (34). The distinct features of the conformational changes of RSK2^NTKD from the AMP-PNP-bound form (33) were described previously (21). In the docking model of IBC-RSK2^NTKD, IBC formed two hydrogen bonds with RSK2, with one involved in the backbone amide group of the hinge loop residue Leu150, and the other engaged with the backbone carbonyl oxygen of the residue Glu197 (Fig. 1B-d). IBC also developed π-interactions between the benzene rings with the gate residue Phe79 in the glycine-rich loop (Fig. 1B-d) and Phe212 in the DFG motif of the activation loop (Fig. S1B). Additionally, the 3-methyl-2-butene tail of the ligand located inside a hydrophobic pocked formed by the residues of Ile50, Leu155, Phe212 and Leu214, while the other carbon atoms of IBC also created hydrophobic interactions with the side-chains of the residues Phe79, Val82, Ala98, Leu102, Val131, Leu145, Leu147, Phe149, Leu150, Leu155, Leu200 and Phe212 around the ATP binding site (Fig. S1B). The docking energy between this compound and RSK2 C-terminal kinase domain (CTKD) was demonstrated to be >2.0 kcal/mol higher, compared with between IBC and RSK2^NTKD from virtual screening and induced fit docking (Table SI). Thus, it was considered that this ligand could be primarily bound inside the ATP binding pocket in the N-terminal kinase domain of RSK2. Finally, these computational results indicate that IBC possibly exhibits ATP-competitive inhibitory effects on ERK1/2 and RSK2 kinases.

In view of the computer predication that IBC may be a potential inhibitor of ERKs/RSK2 signaling, the cytotoxic effects of IBC on liver cancer cells and immortalized hepatocytes were investigated. As depicted in Fig. 2A, IBC significantly decreased the viability of liver cancer HepG2 and Hep3B cells in a concentration- and time-dependent manner. The IC₅₀ values of IBC on HepG2 and Hep3B cells at 48 h were 16.45 and 13.22 µM, respectively. In contrast, a less significant cytotoxic effect was observed in immortalized normal liver L02 cells. The EdU incorporation assay was performed to further determine the effects of IBC on the proliferation of HepG2 and Hep3B cells. As depicted in Fig. 2B, following exposure to various concentrations of IBC for 48 h, the ratio of EdU-positive cells was gradually reduced with the increasing concentrations of IBC. Furthermore, the colony-forming ability of HepG2 and Hep3B cells was attenuated by IBC in a concentration-dependent manner (Fig. 2C). Collectively, these observations indicated that IBC effectively suppresses the viability and proliferation of liver cancer cells rather than normal hepatocytes.

The effects of IBC on cell cycle distribution and apoptosis were further analyzed using flow cytometry. Notably, it was determined that IBC exerted no notable influence on cell cycle distributions of HepG2 and Hep3B cells, even up to the concentration of 20 µM (Fig. S2). However, treatment with IBC could induce a notable concentration-dependent increase of cell apoptosis, particularly early apoptosis, in HepG2 and Hep3B cells (Fig. 3A). Subsequently, the effect of IBC on apoptosis-associated molecules was investigated. As depicted in Fig. 3B, exposure of HepG2 cells to IBC caused the downregulation of MDM2 and Bcl-2, along with the increase of p53 and Bax, in a concentration-dependent manner. Additionally, procaspase-9 exhibited a significant decrease response to IBC treatment, concomitant with the increasing levels of caspase-3 and −7 activities and cleavage of PARP (Fig. 3C), indicating a hierarchical activation of the apoptotic caspase cascade. Collectively, these results indicated that the induction of caspase-mediated apoptosis was involved in the antitumor activity of IBC on liver cancer cells.

It has been determined from computer-aided virtual screening and molecular modeling that ERK1/2 and RSK2 may represent potential targets of IBC. To further determine whether IBC directly binds with ERK1/2 and RSK2, an in vitro pull-down assay using HepG2 or Hep3B cell lysates was conducted. As depicted in Fig. 4A, ERK1/2 and RSK2 could bind to the IBC-Sepharose 4B beads, but not to DMSO-Sepharose 4B beads. Additionally, IBC could not selectively bind to JNK/SAPK (Fig. 4A).

Subsequently, an IP kinase assay was conducted to investigate the effect of IBC on RSK2 kinase activity. Histone H3 is a well-known phosphorylation substrate of RSK2 (35). The full-length RSK2 was transfected into HepG2 cells and RSK2 protein was immunoprecipitated by Flag-tagged antibody. The precipitates were subjected to an in vitro kinase assay with various concentrations of IBC and histone H3 peptide as a substrate. As depicted in Fig. 4B, treatment with IBC inhibited the levels of histone H3 phosphorylation at Ser10 in a concentration-dependent manner, compared with untreated control, indicating that IBC could effectively block RSK2 kinase activity in vitro. Additionally, whether IBC interferes with the activity of endogenous RSK2 in HepG2 cells was investigated. Cells were treated with various concentrations of IBC for 2 h, followed by stimulation with EGF. The results from RSK2 IP kinase assay revealed that IBC also significantly inhibited EGF-induced endogenous RSK2 kinase activity (Fig. 4C). Overall, these observations indicated that IBC could effectively suppress the RSK2 activity by directly binding to ERK1/2 and RSK2 in liver cancer cells.

Based on the aforementioned experimental results, it was concluded that IBC exerted antitumor activity on liver cancer cells through regulating the ERKs/RSK2 signaling pathway. The effects of IBC on EGF-induced phosphorylation of ERK1/2 and RSK2 were examined by western blotting. As expected, EGF, a tumor promoter, induced the phosphorylation of ERK1/2 and RSK2 in HepG2 and Hep3B cells (Fig. 5A). Treatment with IBC significantly inhibited EGF-induced phosphorylation of RSK2 in a concentration-dependent manner (Fig. 5A), consistent with the decrease of endogenous RSK2 activity by IBC. However, the EGF-induced phosphorylation of ERK1/2 does not appear to be affected by IBC (Fig. 5A). To demonstrate the inhibitory role of IBC on the ERKs/RSK2 signaling pathway, the status of its downstream target proteins, including CREB, ATF1 and histone H3, whose phosphorylation is increased in response to EGF-induced ERKs/RSK2 activation, were evaluated. As depicted in Fig. 5B, the phosphorylation levels of CREB, ATF1 and histone H3 induced by EGF were notably abrogated by IBC in a concentration-dependent manner. AP-1 is a dimeric transcription factor composed of Jun, Fos or ATF protein family members (36). It is well known that RSK2 is involved in the regulation of AP-1 transcriptional activity through its phosphorylation of histone H3 at Ser10 (31). The present results indicated that EGF promoted AP-1 transcriptional activity and that treatment with IBC resulted in a concentration-dependent inhibition of AP-1 transactivation in liver cancer cells (Fig. 5C). Collectively, these results provided evidence that IBC could effectively block the ERKs/RSK2 downstream signaling pathway, which may be responsible for the anti-proliferation activity of IBC to some extent.

To further determine the role of RSK2 in IBC-induced suppression of liver cancer cells, the expression levels of RSK2 in HepG2, Hep3B and L02 cells were detected. As depicted in Fig. 6A, compared with L02 cells, elevated total- and phospho-RSK2 protein levels were observed in HepG2 and Hep3B cells. On this basis, a vector encoding RSK2 was transfected into L02 cells, and then cell viability was evaluated with a CCK-8 assay under the conditions with or without IBC. Expectedly, overexpression of RSK2 significantly promoted the proliferation of L02 cells (Fig. S3). Furthermore, enforced RSK2 expression in L02 cells notably enhanced the inhibitory effect of IBC on cell viability (Fig. 6B). Additionally, it was observed that RSK2 overexpression notably increased colony formation capacity in L02 cells, whereas IBC effectively counteracted the effect of RSK2 on inducing colony formation (Fig. 6C). Conversely, RSK2 knockdown in HepG2 cells reduced the effect of IBC on suppressing cell proliferation in a concentration-dependent manner, compared with the control cells (Fig. 6D). Overall, these results supported the assertion that the RSK2-mediated signaling pathway serves a critical role in the inhibitory effect of IBC on liver cancer cells.