Curcumin, resveratrol and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) and 0.25% (w/v) Trypsin-EDTA were obtained from Gibco-BRL (Gaithersburg, MD, USA). The MTT cell proliferation and cytotoxicity assay kit, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), N-acetylcysteine (NAC), caspase-3, -8 and -9 colorimetric assay kits, and Hoechst 33258 were obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Z-VAD-FMK was obtained from R&D Systems (Minneapolis, MN, USA). Propidium iodide (PI) and Annexin V-FITC were purchased from BD Pharmingen (Minneapolis, MN, USA). Antibodies against XIAP and survivin were purchased from Bioworld Technology Co., Ltd. (St. Louis Park, MN, USA). Antibodies against β-actin were from Cell Signaling Technology (Danvers, MA, USA).

Murine hepatocarcinoma cell line Hepa1-6 was purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and was grown in DMEM with 10% FBS and 1% penicillin/streptomycin, and maintained at 37°C in a humidified incubator with a 5% CO₂ atmosphere. Exponentially growing Hepa1-6 cells were seeded into desired plates and allowed to attach for 24 h before treatment. Cells were treated with various doses of curcumin and resveratrol dissolved in DMSO. The identical volume of DMSO was used as a control.

The effects of curcumin and resveratrol on cell proliferation were detected by MTT assay. Briefly, Hepa1-6 cells were seeded into 96-well plates (3.5×10³ cells/well) and allowed to attach for 24 h before treatment. The cells were exposed to various doses of curcumin and/or resveratrol for 48 h, and cell viability was evaluated every 24 h by MTT assay according to the manufacturer’s instructions. The cell survival rate was calculated as follows: Cell survival rate (%) = experimental OD value/control OD value × 100.

Morphological changes characteristic of apoptosis were detected by Hoechst 33258 staining. Briefly, 6×10⁴ Hepa1-6 cells were seeded into 6-well plates and incubated for 24 h. Hepa1-6 cells were treated with curcumin and/or resveratrol for 48 h, and stained with Hoechst 33258 for 5 min at room temperature. The cells were observed and photographed using an inverted fluorescence microscope (AFM010-2, Nikon, Japan).

Hepa1-6 cells were treated as indicated, collected, and stained with Annexin V-FITC and PI as recommended by the manufacturer. Apoptotic cells were detected by flow cytometry, and the extent of apoptosis was calculated with FlowJo software (version 7.6.1).

Intracellular ROS production was detected by DCFH-DA staining. DCFH-DA is cleaved intracellularly by non-specific esterases to form DCFH, which is further oxidized by ROS to form the fluorescent compound DCF (22). Briefly, Hepa1-6 cells (1×10⁵) were placed in 60-mm culture dishes. On the second day, cells were exposed to the indicated concentrations of agents for a 48 h treatment, and stained with DCFH-DA (10 μM) at 37°C for 20 min. The stained cells were collected and added to black 96-well plates at a density of 2×10⁵ cells/well. The presence of DCF fluorescence was quantified with a fluorescence microplate reader (Thermo Scientific Varioskan Flash; Thermo Fischer Scientific, Waltham, MA, USA) at wavelengths of 488 nm for excitation and 525 nm for emission. For ROS inhibition, cells were pretreated with NAC (2.5 mmol/l for 2 h), followed by curcumin and/or resveratrol treatment.

After treatment with curcumin and/or resveratrol, caspase-3, -8 and -9 activities were measured by the cleavage of the specific chromogenic substrate according to the manufacturer’s instructions. For inhibiton of caspase activity, Hepa1-6 cells were pretreated with Z-VAD-FMK (50 μmol/l for 2 h) and further treated with curcumin and/or resveratrol.

Western blot analysis was performed as previously described (23,24). Briefly, after drug treatment, Hepa1-6 cells were collected, lysed and subjected to 6–12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel, and transferred to nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). The transferred membranes were blocked with 5% non-fat milk, washed and probed with the indicated antibodies. The specific antigen-antibody complex was visualized using an enhanced chemiluminescence detection method (Amersham, Buckinghamshire, UK).

Data are expressed as the means ± standard deviation of at least two independent experiments, each conducted in triplicate. Statistical significances between the control and drug treatment were determined by one-way ANOVA. A value of P<0.05 was considered to indicate a statistically significant result.

The antiproliferative effects of curcumin and resveratrol on hepatocarcinoma Hepa1-6 cells were assessed with the MTT assay. As shown in Fig. 1, curcumin (5–40 μM) and resveratrol (10–160 μM) significantly inhibited the proliferation of Hepa1-6 cells in a dose- and time-dependent manner (P<0.05).

To assess the combined effects of curcumin and resveratrol on Hep1-6 cells, we treated Hepa1-6 cells with curcumin, resveratrol or both agents in a constant ratio to one another. As shown in Fig. 2A, the combination treatment of curcumin and resveratrol was more effective in inhibiting the proliferation of Hepa1-6 cells when compared with the treatment of either agents alone (P<0.01), which indicated an interaction between the two drugs. The precise nature of this interaction was further analyzed by the median-effect method (25), where the combination indices (CI) of less than, equal to, and more than 1 indicate synergistic, additive and antagonistic effects, respectively. The CI value was <1 for the combination treatment when the proliferation inhibition was >20%, indicating a synergistic effect between curcumin and resveratrol on Hepa1-6 cell proliferation (Fig. 2B). Based on these observations, we selected 10 μM of curcumin and 40 μM of resveratrol (~50% inhibitory effect for the combination treatment) to carry out the subsequent studies.

To assess the effects of curcumin and resveratrol on apoptosis, Hepa1-6 cells were treated with curcumin and/or resveratrol and subjected to Hoechst 33258 staining to visualize the apoptotic morphological alterations. As shown in Fig. 3A, following treatment with curcumin and/or resveratrol for 48 h, a portion of Hepa1-6 cells exhibited nuclear shrinkage or fragmentation, indicating the occurrence of apoptotic processes. Flow cytometric analysis was used to quantify the extent of apoptosis. As shown in Fig. 3B and C, curcumin and resveratrol significantly induced apoptosis of the Hepa1-6 cells (P<0.01). Combination treatment of curcumin and resveratrol was found more effective in inducing apoptosis than either agent alone (P<0.01).

Cell apoptosis is executed by a caspase (cysteine aspartate-specific proteinase) cascade, and activation of caspases has been recognized as a hallmark of apoptosis (26). We further observed the effects of curcumin and/or resveratrol on caspase activation in Hepa1-6 cells. As shown in Fig. 4A-C, curcumin and/or resveratrol treatment significantly activated caspase-3, -8 and -9 in Hepa1-6 cells (P<0.01). Combination treatment of curcumin and resveratrol significantly enhanced caspase-3, -8 and -9 activation when compared with that following treatment with either agent alone (P<0.01). In addition, Z-VAD-FMK, a pan caspase inhibitor, completely inhibited curcumin and resveratrol-induced apoptosis in Hepa1-6 cells (P<0.01) (Fig. 4D). These results suggest that caspase-3, -8 and -9 activation may be an important mechanism involved in the apoptosis of Hepa1-6 cells induced by the combination treatment of curcumin and resveratrol.

It has been confirmed that excess ROS promotes cell death, and both curcumin and resveratrol upregulate ROS to induce apoptosis in cancer cells (27–29). Thus, we further examined the effects of the combination treatment of curcumin and resveratrol on ROS production in Hepa1-6 cells by DCFH-DA staining. As shown in Fig. 5, curcumin and/or resveratrol treatment resulted in significant ROS generation as indicated by brightly green fluorescence in the Hepa1-6 cells (P<0.01). Combination of curcumin and resveratrol significantly enhanced the ROS generation when compared to that following treatment with either agent alone (P<0.01).

To elucidate the effects of ROS on curcumin and resveratrol-induced apoptosis, we further evaluated cell apoptosis after NAC (ROS scavenger) pretreatment by flow cytometric analysis. When compared without the blocking of NAC, apoptosis induced by curcumin and/or resveratrol was partially but significantly abrogated by NAC pretreatment (P<0.05) (Fig. 6A). Particularly in the combination treatment group, the percentage of apoptosis was reduced to almost half of the apoptosis noted in the absence of NAC. In addition, curcumin and/or resveratrol-induced activation of caspase-3, -8 and -9 was also reduced by NAC pretreatment, particularly in the combination treatment group (P<0.05) (Fig. 6B-D). These observations suggest that the combination treatment of curcumin and resveratrol induced apoptosis, and caspase activation was associated with ROS production.

It has been reported that the inhibitors of apoptosis proteins, such as XIAP and survivin, are abnormally expressed in hepatocarcinoma cells and they have been confirmed as therapeutic targets for natural products (30–32). We further evaluated the effects of the combination treatment of curcumin and resveratrol on XIAP and survivin expression in Hepa1-6 cells. As shown in Fig. 7, combination treatment with curcumin and resveratrol significantly inhibited XIAP and survivin expression in the Hepa1-6 cells, when compared to the expression in the control or following treatment with either agent alone.