Human hepatic carcinoma cell lines HepG2 and SMMC7721, and mouse embryonic liver cell line BNL CL.2 were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (catalog no. 12800-058; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal calf serum (Standard Grade; Lanzhou Bailing Biotechnology Co., Ltd., Lanzhou, China.) at 37°C and 5% CO₂.

Small interfering RNA (siRNA) targeting human RACK1 (5′-GGATGAGACCAACTATGGAAT-3′) and human CBR1 (5′-ATACGTTCACCACTCTCCCTT-3′) and small hairpin RNA targeting mouse RACK1 (5′-GTCCCGAGACAAGACCATAAA-3′) were designed and chemically synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). The siRNAs were delivered into the SMMC7721 cells at 50% confluence using Lipofectamine^® RNAiMAX transfection reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells were transfected for 48 h and then analyzed for various parameters. SMMC7721 stable clones, which were a gifts from Dr Wendie Wang (Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing, China), were selected in 600 µg/ml G418 sulfate (catalog. no. 11811-023; Thermo Fisher Scientific, Inc.) for approximately 2 months.

Cells were harvested and lysed in lysis buffer (20 mM Tris, pH 7.6, 250 mM NaCl, 1% Nonidet P-40, 3 mM EDTA, 1.5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 10 µg/ml aprotinin, 10 mM p-nitrophenylphosphate, 1 mM Na₃VO₄, 1 mM dithiothreitol). Following clarification by centrifugation at 5,000 × g for 15 min at 4°C, cell lysates were incubated with the indicated antibodies in the presence of 30 µl [50% (v/v)] of protein A-Sepharose beads (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA) at 4°C for 4 h. Precipitates were washed with washing buffer [20 mM Tris (pH 7.6), 250 mM NaCl, 1% Nonidet P-40, 3 mM EDTA, 1.5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid and 1 mM phenylmethane sulfonyl fluoride) at least three times. For western blot analysis, cell lysates or co-IP samples underwent 12% SDS-PAGE for 2 h, followed by transferal to polyvinylidene difluoride membranes for 3 h and blocking with 5% nonfat milk in TBS containing 0.1% Tween-20 (TBST) for 1 h at room temperature. Membranes were incubated with primary antibodies against RACK1 (catalog no. 610177; BD Biosciences, San Jose, CA, USA), CBR1 (catalog no. ab4148; Abcam, Cambridge, MA, USA), β-actin (catalog no. 47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (catalog no. sc-81545; Cell Signaling Technology, Inc., Danvers, MA, USA) at a dilution of 1:1,000 overnight at 4°C. Following three times washing with TBST (10 min each wash), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:5,000 for 1 h at room temperature (polyclonal goat anti-rabbit or goat anti-mouse secondary antibodies; catalog no. ZB2301 and ZB2305, respectively; OriGene Technologies, Inc., Beijing, China), followed by additional washing. The membranes were subjected to exposure in the dark and the immunoreactive bands were visualized with an enhanced chemiluminescence kit (GE Healthcare Life Sciences, Chalfont, UK). Quantification of western blot were performed by using Gel-Pro Analyzer 4.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Cells treated with 10 ng/ml TNF-α (R&D Systems, Inc., Minneapolis, MN, USA) with or without 10 µg/ml CHX (Sigma-Aldrich; EMD Millipore), or 1 mM H₂O₂ for 24 h were digested by 0.25% trypsin for approximately 2 min with gentle shaking, and subsequently harvested. Following washing twice with PBS, the cell pellet was resuspended in 200 ml PBS containing Annexin-V and propidium iodide (PI)/7-aminoactinomycin D (BD Biosciences) and incubated at 4°C for 30 min, followed by flow cytometry assay.

Cells were resuspended and incubated in pre-warmed Hank's balanced salt solution (HBSS) containing 10 mM carboxy-.2′,7′-dichlorodihydrofluorescein diacetate (Thermo Fisher Scientific, Inc.) for 30 min at 37°C, followed by incubation with 10 ng/ml TNF-α or 400 µmol/l H₂O₂ for 30 min at 37°C. Cells were washed with HBSS twice and subjected to flow cytometry.

Cell death assay experiments were performed independently at least three times. Statistical differences between groups were assessed by Students t-test. Descriptive statistics were computed by using Excel 2007 (Microsoft Corporation, Redmond, WA, USA). P<0.05 was considered to indicate a statistically significant difference.

The present study initially investigated the correlation between RACK1 and TNF-α-induced cell death in SMMC7721 cells. SMMC7721 cells were transiently transfected with RACK1 siRNA or NC siRNA by using the iMAX delivery system. A total of 48 h later, the cells were treated with 10 ng/ml TNF-α with or without 10 µg/ml CHX for 24 h, followed by cell death assay with Annexin-V and PI double staining. Transfection with RACK1 siRNA dramatically decreased RACK1 protein levels (Fig. 1A), leading to an increased cell death rate compared with NC siRNA following co-treatment with TNF-α and CHX (P=2.67×10⁻⁷; Fig. 1B). Notably, treatment with TNF-α alone only caused slight cell death, and cell death was markedly induced in the presence of CHX, a pan-protein synthesis inhibitor (Fig. 1B), indicating that CHX sensitized SMMC7721 cells to TNF-α-induced cell death. It was subsequently investigated whether RACK1 affected ROS responses to stimuli, including TNF-α or hydrogen peroxide. As demonstrated in Fig. 1C, knockdown of RACK1 resulted in increased ROS generation in SMMC7721 cells upon TNF-α and hydrogen peroxide treatment. Taken together, the results of the present study suggested that RACK1 acted as a ROS suppressor to antagonize TNF-α-induced cell death in SMMC7721 cells.

Although it was observed that knockdown of endogenous RACK1 promoted ROS generation upon TNF-α and hydrogen peroxide stimulation, the molecular mechanism(s) by which RACK1 affected ROS remain to be elucidated. Subsequently, a co-IP was performed, followed by mass spectrometry analysis (performed by National Center of Biomedical Analysis, Beijing, China) to identify RACK1-interacting partners (data not shown). Among the candidates of RACK1-interacting proteins obtained, CBR1 was notable due to its close association with ROS. CBR1 was considered to be a ROS suppressor, as it had been reported to protect cells from cytotoxic drug-triggered cell death in doxorubicin-treated HCC cells and As₂O₃-treated leukemia cells (4,18). Therefore, the functions of CBR1 were investigated in TNF-α-treated SMMC7721 cells. As expected, transfection with CBR1 siRNA markedly decreased the protein level of CBR1 (Fig. 2A), leading to increased percentages of cell death induced by TNF-α (P=0.046) and TNF-α/CHX (P=0.00067; Fig. 2B). Subsequently, the present study employed RACK1 stably silenced single clones screened from SMMC7721 cells to investigate whether CBR1 affected the enhanced cell death caused by RACK1 knockdown. As expected, overexpression of green fluorescent protein (GFP)-CBR1 reversed the enhanced cell death in the RACK1 stably silenced clone, at least partially, compared with the clone overexpressing GFP (P=0.00071; Fig. 2C). Cell death upon hydrogen peroxide stimulation was also investigated in HCC cells. Rather than apoptotic cell death, hydrogen peroxide stimulation caused a more necrotic cell death than TNF-α/CHX stimulation (data not shown). However, hydrogen peroxide caused ~80% cell death, and knockdown of CBR1 elevated cell death up to >90% (P=6.42×10⁻⁵; Fig. 2D). Thus, the results of the present study suggested RACK1 may exert its protecting function via CBR1.

To address whether RACK1 affected the functioning of CBR1, it was necessary to address whether and how RACK1 regulates CBR1. Initially, the present study briefly addressed the interaction between RACK1 and CBR1 in vivo. As shown in Fig. 3A, when endogenous CBR1 was immunoprecipitated from SMMC7721 cells with CBR1 antibody, RACK1 was detected in the precipitant, suggesting that RACK1 was able to interact with CBR1 in SMMC7721 cells (Fig. 3A). Furthermore, in SMMC7721 RACK1 stably silenced single clones, CBR1 protein levels exhibited a marked decrease compared to control clones (Fig. 3B), suggesting that RACK1 potentially regulated the protein stability of CBR1. For further investigation, the present study examined the degradation of CBR1 in HCC cells in RACK1 stably silenced clones and control clones upon CHX treatment for various time courses. CBR1 protein exhibited a decrease at 16 and 20 h subsequent to CHX treatment in HCC clones with RACK stable knockdown but exhibited no change in control HCC clones (Fig. 3C). It has previously been reported that RACK1 exhibited higher expression in HCC cells compared to ‘normal’ hepatocytes (10). As expected, an increased level of RACK1 protein was detected in SMMC7721 cells and HepG2 cells, as compared to BNL CL.2 mouse embryonic liver cells (Fig. 3D). Consistent with the idea that RACK1 promotes the stability of CBR1 protein, HepG2 cells exhibited an increased level of CBR1 protein compared with BNL CL.2 cells (Fig. 3E). This finding is consistent with a previous report in which CBR1 demonstrated overexpression in 56 (72%) out of 78 human HCC tissues (6). Notably, upon TNF-α/CHX stimulation, BNL CL.2 cells exhibited a marked increase in cell death compared to HepG2 cells (P=0.00017; Fig. 3F). These results indicate that upregulation of RACK1 together with CBR1 may have a significant role in the transformation of normal liver cells to malignant cells.