Sodium cantharidinate, Hoechst 33258, MTT and RNase were purchased from Sigma (St. Louis, MO, USA). Z-DEVD-FMK (HY-12466, Caspase-3 inhibitor) was purchased from MedChem Express (Monmouth Junction, NJ, USA). Propidium iodide and Annexin V-FITC Apoptosis Detection kit was purchased from BD Biosciences (San Jose, CA, USA). LC3 siRNA reagent kit was purchased from Cell Signaling Technology (Danvers, MA, USA). Dulbecco's modified Eagle's medium (DMEM), trypsin, fetal bovine serum (FBS), PBS, penicillin and streptomycin were obtained from Gibco BRL Life Technologies (Grand Island, NY, USA).

Sodium cantharidinate was dissolved in PBS (pH 7.2) to prepare a stock solution at a concentration of 1.0 mM which was stored at −20°C. DMEM complete medium was added to dilute the sodium cantharidinate to the appropriate concentrations prior to use.

HepG2 cells were routinely cultured in DMEM complete medium which contains 50 U/ml antibiotics and 10% fetal bovine serum (FBS) under the conditions of 5% CO₂ at 37°C in cell incubator (HERAcell 150i; Thermo Fisher Scientific, Waltham, MA, USA). Following trypsinization to passage the cells in 75 T flask 3–5 days, the cells were counted and reseed in 96-well plate in DMEM complete medium without or with sodium cantharidinate for MTT array or apoptosis detection.

The inhibitory effect of sodium cantharidinate on the proliferation of HepG2 cells were detected via MTT assays. All experiment steps were performed following the instructions of the kit. Briefly, the cells were seeded on 96-well plates at a density of 5×10⁴/ml at a volume of 200 µl per well. All groups without or with sodium cantharidinate (0, 2.0, 5.0, 12.5 µM) were incubated 6–24 h. MTT (1.0 mg/ml) was added to each well, and the cells were incubated for 4 h. The MTT solution was then aspirated, and 100 µl DMSO was added. The 96-well plates were read using a microplate spectrophotometer (Synergy H1, BioTek, Winooski, VT, USA) at 540 nm. The experiments were repeated in triplicate. The inhibition percentage was calculated as (1 - the value in experimental group / the value in the control group) ×100%.

Annexin V-FITC and PI double staining flow cytometry analyses were employed. The HepG2 cells were plated in 96-well plates containing 200 µl medium at a density of 5×10⁴ cells/well. The induction of apoptosis in the HepG2 cells were examined without or with sodium cantharidinate (5.0 µM). After culture, the cells were collected in 1.5 ml centrifuge tubes, washed three times with cold PBS and binding buffer, and then stained with Annexin V-FITC and PI (Annexin V-FITC Apoptosis Detection kit; BD Biosciences) for apoptosis detection. Briefly, HepG2 cells in centrifuge tubes were re-suspended in binding buffer. Then, 5 µl of FITC-Annexin V was added to the tubes, which were incubated for 10 min followed by the addition of 5 µl PI. The samples were then incubated with PI for another 15 min and immediately analyzed using a flow cytometer (FACScan; BD Biosciences) with the Flowjo FACS analysis software. The cells in the different portions represented the different cell states as follows: the late-apoptotic cells were present in the upper right portion, the viable cells were present in the lower left portion, and the early apoptotic cells were present in the lower right portion.

HepG2 cell lysates were separated by SDS-PAGE under non-reducing conditions on a 10% polyacrylamide gel. The proteins were then transferred onto PVDF membranes (GE Healthcare Life Sciences, Piscataway, NJ, USA) by electroblotting. The membranes were blocked with blocking buffer overnight at 4°C and then incubated with the caspase-3, cleaved caspase-3, LC3-I and LC3-II antibodies for 1.5 h at room temperature. The membranes were then washed by washing buffer six times and incubated with HRP-conjugated secondary antibodies for 1 h. After washing, protein bands were visualized using an enhanced chemiluminescent system (Thermo Fisher Scientific). The primary antibodies used (caspase-3, cleaved caspase-3, LC3-I, LC3-II and β-actin) were all obtained from Cell Signaling Technology.

Hoechst 33258 (Sigma) were used to assessed apoptotic nuclear changes. After treatment with 5.0 µM sodium cantharidinate for 0, 6, 12, and 24 h, cells were fixed with 4% paraformaldehyde, stained with Hoechst 33258 (2 µg/ml) for 30 min, washed in PBS, and examined using Olympus FV1000 confocal laser microscopy to reveal cell chromatin condensation. Briefly, HepG2 cells were cultured on coverslips overnight, then treated with 5.0 µM sodium cantharidinate for 6 h and rinsed with PBS at least three times. Cells were fixed for 20 min with 4% paraformaldehyde after incubation, then permeabilized with 0.1% Triton X-100 for 5 min, finally blocked with bovine serum albumin. Then incubated with primary antibodies against LC3 (1:50 dilution) overnight at 4°C, then in FITC/Rhodamine Red-conjugated secondary antibodies (1:400 dilution) (all antibodies, Santa Cruz Biotechnology, CA, USA) for 0.5 h, and stained with Hoechst 33258 (2 µg/ml) for 2 min, washed with PBS three times, and examined by confocal fluorescence microscopy.

All data were analyzed and assessed for significance using the Pearson omnibus normality test. All data are presented as the mean ± the standard error of the mean. Mean values were compared using paired t-tests (two groups) followed by the Bonferroni correction for multiple comparison tests. p-values <0.05 were considered to indicate a statistically significant result. All statistical tests were performed with prism software (GraphPad, San Diego, CA, USA).

HepG2 cells were treated with different doses of sodium cantharidinate for different time intervals, and MTT results showed that sodium cantharidinate effectively inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner (Fig. 1A). We then selected 5.0 µM sodium cantharidinate for treatment of HepG2 cells at different time intervals, and the apoptotic effector caspase-3 was detected by western blotting. The results showed that the expression of cleaved caspase-3 was increased at all three time points (4-fold change, p<0.01, Fig. 1B and C). The cell nucleus stained with Hoechst 33258 was observed using confocal laser scanning microscopy. The results suggested that sodium cantharidinate induced apoptosis in HepG2 cells, which keeps consistency to the MTT array (Fig. 1D). Annexin V-FITC and PI double staining assay was also performed to confirm the cytotoxicity of sodium cantharidinate on HepG2 cells (Fig. 1E and F). The results showed that comparing with the control group, the numbers of early and late apoptotic cells increased significantly in the treated group. The proportion of early and late apoptotic cells in the sodium cantharidinate treatment group reached 37.2%, which was greater than the proportion observed in the control group (10.1%, p<0.01). To determine the effect of caspase-3 on the sodium cantharidinate inducement of HepG2 cell apoptosis, a caspase-3 inhibitor, Z-DEVD-FMK (100 µM) was added to the well, or not. Cells were treated by sodium cantharidinate for 6 h, then apoptosis was determined by FACS. The proportion of apoptotic cells decreased from 35.2% to 17.8% when the Z-DEVD-FMK was added (Fig. 1G, p<0.01). This finding indicated that sodium cantharidinate significantly induced HepG2 cell apoptosis by caspase-3 activity.

Studies suggest that autophagy may be involved in the antitumor effect of drugs (25). Therefore, we analyzed the protein expression of the autophagic maker protein LC3 in response to 5.0 µM sodium cantharidinate by western blotting. The results showed that the protein expression ratio of LC3-II and LC3-I was significantly increased by sodium cantharidinate treatment for 6–24 h (Fig. 2A and B). Furthermore, indirect immunofluorescence showed that LC3 had translocated to the cytoplasm, forming punctate aggregates, and the fluorescence intensity of LC3 was also enhanced (Fig. 2C), suggesting that sodium cantharidinate induced autophagy in HepG2 cells. The expression of LC3 expressed in HepG2 cells was analyzed by flow cytometry. The results are shown in Fig. 2D and E. The LC3 expression level was much higher in HepG2 cells treated with sodium cantharidinate than controls (MFI: 2508±165 vs. 1458±89, p<0.001). These results showed that sodium cantharidinate induced HepG2 cell autophagy through LC3 pathway.

Previous research used 3-MA to inhibit autophagy and prove that autophagy is involved in the growth inhibition of hepatoma cells (26). Therefore, we combined 5 mM 3-MA and 5 µM sodium cantharidinate treatment in HepG2 cells for 6 h, and detected the protein expression of LC3-II and LC3-I by western blotting. Sodium cantharidinate combined with 3-MA resulted in a reduction of protein expression ratio of LC3-II and LC3-I compared with sodium cantharidinate alone (3-fold change, p<0.01, Fig. 3A and B). Indirect immunofluorescence demonstrated that LC3 was distributed in both the cytoplasm and the nucleus, and the fluorescence intensity was significantly reduced (Fig. 3C), showing that 3-MA inhibited sodium cantharidinate-induced autophagy effectively. Furthermore, Annexin V-FITC and PI double staining assay was also performed to confirm the LC3 inhibitor influences the cytotoxicity of sodium cantharidinate on HepG2 cells (Fig. 3D). The results showed that compared with the control group, the numbers of early and late apoptotic cells decreased significantly when 3-MA was combined with sodium cantharidinate. The proportion of early and late apoptotic cells in the sodium cantharidinate treatment group reached 37.2%, but it decreased to 22.2% when 3-MA was added (p<0.01).

We applied RNAi technology to inhibit LC3 expression on HepG2 cells. After the LC3 siRNA treatment of HepG2 cells for 24 h, we observed the expression rate of green fluorescent protein (GFP) to be >85% by fluorescence microscopy (Fig. 4A). LC3 protein expression was significantly decreased as detected by western blotting (p<0.001, Fig. 4B and C) and qRT-PCR (p<0.01, Fig. 4D). FACS analysis showed that the MFI level of LC3 was also significantly decreased (p<0.001, Fig. 4E and F). Then, Annexin V-FITC and PI double staining assay was also performed to confirm that the LC3 gene was silenced after sodium cantharidinate treatment. The results showed that compared with the control group, the numbers of early and late apoptotic cells decreased significantly when sodium cantharidinate treatment of the LC3 silenced HepG2 cells wre compared to the control cells (p<0.001, Fig. 4G and H). These results showed that LC3 autophagy pathway played an important role in the sodium cantharidinate induced HepG2 cell apoptosis.