Sorafenib (Nexavar) and wogonin were purchased from Bayer AG (Leverkusen, Germany) and Sigma-Aldrich (Merck Millipore, Darmstadt, Germany), respectively. Antibodies against poly (ADP-ribose) polymerase (PARP) (catalog no. 556494) and p62 (catalog no. 610832) were acquired from BD Biosciences (Franklin Lakes, NJ, USA). Anti-β-actin (catalog no. 60008–1-Ig) and anti-light chain 3 (LC3)B antibodies (catalog no. L7543) were obtained from ProteinTech Group, Inc. (Chicago, IL, USA) and Sigma-Aldrich (Merck Millipore), respectively. Chloroquine (CQ) and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (Merck Millipore). The pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl (Z-VAD) was obtained from Calbiochem (Merck Millipore).

The human HCC cell lines Hep3B, Bel-7402, HepG2 and SMMC-7721 were purchased from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). These cells were cultured in Dulbeccos modified Eagles medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck Millipore), 100 U/ml penicillin and 100 µg/ml streptomycin under standard incubation conditions (37°C and 5% CO₂).

Following treatment, cell death was quantitatively detected by a cytotoxicity assay based on the release of LDH using a cytotoxicity detection kit (Promega Corporation, Madison, WI, USA) as described previously (34). All the experiments were repeated 3–5 times and data were expressed as the mean ± standard deviation (SD). Flow cytometry was applied to detect apoptosis in cultured cells by using an Annexin V-FITC Apoptosis Detection kit purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Cells were double stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). Apoptosis was then analyzed by flow cytometry (BD Biosciences). Early apoptotic cells with exposed phosphatidylserine but intact cell membranes bind Annexin V-FITC but exclude PI, and will be reported in the lower right-hand quadrant (B4). Necrotic or apoptotic cells in terminal stages will be both Annexin V-FITC and PI positive, and will be reported in the upper right-hand quadrant (B2).

Whole cell lysates were prepared by lysing cells in M2 buffer [20 mmol/l Tris-HCl (pH 7.6), 0.5% NP40, 250 mmol/l NaCl, 3 mmol/l EDTA, 3 mmol/l ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid, 2 mmol/l dithiothreitol, 0.5 mmol/l phenylmethylsulfonyl fluoride, 20 mmol/l β-glycerophosphate, 1 mmol/l sodium vanadate and 1 µg/ml leupeptin]. The concentration of proteins in the cell lysates was quantified by Bio-Rad Protein Assay (Bio Rad Laboratories, Inc., Hercules, CA, USA). Cell lysates (~50 µg) were resolved by SDS-PAGE ((8% for detecting PARP, 10% for detecting p62 and β-actin, and 15% for detecting LC3B), transferred to a polyvinylidene fluoride membrane and detected with various antibodies: Anti-PARP (1:500; overnight at 4°C); anti-p62 (1:1,000; overnight at 4°C); anti-β-actin (1:5,000; 1 h at room temperature); and anti-LC3B (1:1,000; overnight at 4°C). Subsequently, the membrane was washed three times for 5 min each with TBS containing Tween-20. Next, peroxidase-conjugated goat anti-mouse immunoglobulin (Ig)G (catalog no. ZB 2305; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) and peroxidase-conjugated goat anti-rabbit IgG (catalog no. ZB 2301; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) were added at a 1:5,000 dilution and incubated with the membrane at room temperature for 30 min. The specific proteins were visualized by enhanced chemiluminescence (EMD Millipore, Billerica, MA, USA) using Image Lab™ station (Bio-Rad Laboratories, Inc.). Each experiment was repeated at ≥3 times and representative results are shown.

Data are expressed as the mean ± SD. Statistical significance was examined by paired Students t test using SPSS version 21.0 software (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate whether wogonin is able to enhance the anticancer activity of sorafenib, Bel-7402 cells were first treated with increasing concentrations of wogonin (10–40 µM) and a fixed concentration of sorafenib (4 µM), and cell death was measured by LDH release assay. The results indicated that, while sorafenib alone caused <20% cell death and wogonin had little cytotoxicity, wogonin sensitized Bel-7402 cells to sorafenib-induced cell death in a dose-dependent manner (Fig. 1A). Approximately 80% of the cells were killed at the highest dose of wogonin used (40 µM), a concentration at which wogonin alone caused little cell death (<10%). The combined cytotoxic effect of wogonin and sorafenib was synergistic, as evaluated by combination index analysis as described previously (35) (Table I). A similar dose-dependent potentiation of cytotoxicity was detected when increasing concentrations of sorafenib (2–4 µM) with a fixed wogonin dose (40 µM) were used (Fig. 1B). The sensitization of sorafenibs anticancer activity by wogonin was validated in other human HCC cell lines. In Hep3B cells, a similar dose-dependent synergism with fixed concentration of either sorafenib or wogonin was observed (Fig. 1C and D). Consistently, wogonin sensitized HepG2 and SMMC-7721 cells to sorafenib-induced cell death (Fig. 1E and F). These results suggest that the combination of wogonin and sorafenib is effective in sensitizing HCC cells to sorafenib-induced cytotoxicity.

As both wogonin and sorafenib can induce apoptosis, it was hypothesized that the enhanced cell death observed in wogonin and sorafenib co-treatment was achieved through potentiation of apoptosis. Hep3B cells were treated with sorafenib in the absence or presence of wogonin, and apoptosis was analyzed by Annexin V-FITC and PI staining followed by flow cytometric assay. Both early (B4) and late (B2) apoptotic cells were significantly increased upon sorafenib and wogonin co-treatment (Fig. 2). The cleavage of the caspase-3 substrate PARP, which is a marker of apoptotic pathway activation, was increased in co-treated Hep3B and Bel-7402 cells, as detected by western blotting (Fig. 3A and B). Additionally, the pan-caspase inhibitor Z-VAD significantly suppressed the enhanced cytotoxicity induced by co-treatment with sorafenib and wogonin in Hep3B and Bel-7402 cells (Fig. 3C and data not shown, respectively). These results suggest that the enhanced cytotoxicity induced by sorafenib and wogonin combination was due to potentiation of apoptosis.

As sorafenib activates autophagy and the latter can either activate or suppress apoptosis, it was further investigated if autophagy is involved in the synergistic cytotoxity of sorafenib and wogonin. Sorafenib induced autophagy, which was shown as increased expression of LC3-II and decreased expression of p62, two autophagy hallmarks (Fig. 4A), in addition to autophagic flux (Fig. 4B). The role of sorafenib-induced autophagy was determined to be cytoprotective, since suppression of autophagy with two different autophagy inhibitors, CQ and 3-MA, remarkably increased sorafenib-induced cell death in both Bel-7402 and Hep3B cells (Fig. 5A and B). Next, the effect of wogonin on sorafenib-induced autophagy was examined. Wogonin effectively inhibited sorafenib-induced autophagy, which was shown as suppression of sorafenib-induced LC3-II expression (Fig. 6). Therefore, these data demonstrate that wogonin likely potentiated sorafenib-induced cell death through inhibiting autophagy.