Sal B was purchased from Sigma (Sigma-Aldrich Corp., St. Louis, MO, USA). 3-MA and CQ were purchased from J&K Chemical Ltd. (J&K Chemical Ltd., Beijing, China). JC-1 and LysoTracker Red were obtained from Invitrogen (Guangzhou, China). The primary antibodies against LC-3, p62, Beclin-1, cleaved PARP, cleaved caspase-3, cytochrome c, total or phospho-AKT (Ser473), total or phospho-mTOR (Ser2448), phospho-4EBP1 (Thr70), and phospho-P70S6K (Thr389) were purchased from Cell Signaling Technology (Boston, MA, USA). The secondary antibodies were HRP-conjugated anti-rabbit IgG purchased from Cell Signaling Technology. The FITC-conjugated anti-rabbit IgG was purchased from Beyotime (Beyotime, Nantong, China).

SK-Hep-1 and Bel-7404 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cell lines were frozen in liquid nitrogen soon after arrival. The experiments with these cells were carried out within 6 generations after resuscitation. SK-Hep-1 and Bel-7404 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal bovine serum (FBS), 100 U penicillin and 100 U streptomycin at 37°C in a humidified incubator of 5% CO₂ and 95% air.

Cell proliferation was determined by MTS assay. Cells were seeded into 96-well plates and treated with Sal B, CQ, 3-MA or a combination. After treatment, 10 μl MTS (Promega) was added into each well for 2-h incubation. The absorbance was measured using a model ELX800 Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) at 490 nm to calculate the proliferation. Three independent experiments were performed to determine the half maximal inhibitory concentration values (IC₅₀).

Cells were incubated at a density of 1,000 cells per well in 6-well plates and treated with a determined dose of Sal B or vehicle control for 2 weeks. After fixation with 4% paraformaldehyde, the colonies formed were counterstained with crystal violet staining solution.

The cell apoptotic rate was determined by flow cytometry analysis using a fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection kit (KeyGen Biotech, Nanjing, China). Cells were collected by trypsinization, washed twice and resuspended in 1X binding buffer at a concentration of 1×10⁶ cells/ml. Then, 100 μl of cells were mixed with 5 μl of FITC Annexin V and 5 μl PI and incubated for 15 min. The samples were sent out for analysis by flow cytometry. The results were analyzed with the BD FACSCalibur™ system.

Cells, seeded at 3×10⁵ into 6-well culture plates, were treated with a determined dose of Sal B for indicated intervals and were incubated with Lyso Tracker for 60 min. Thereafter, cells were washed twice with PBS, followed by fixation in 4% paraformaldehyde and permeabilized with 1% CHAPS buffer (150 mM NaCl, 10 mM HEPES, 1.0% CHAPS) at room temperature for 15 min. Hereafter, cells were incubated with anti-LC3 antibodies for 2 h at 37°C, and incubated with FITC-conjugated anti-rabbit IgG for 1 h at 37°C, and the cell nuclei were stained by DAPI (Invitrogen) for 15 min. Samples were examined under a Zeiss LSM 710 fluorescence microscopy system (Carl Zeiss Inc, Dublin, CA, USA). Images were processed with ZEN LE software. For quantification of LC3-positive cells, 150–200 cells were randomly selected from the acquired images and counted. Cells with more than five dots of specific green or yellow signals were considered to be LC3-positive.

Cells, seeded at 3×10⁵ into 6-well culture plates, were treated with a dose of 200 μM Sal B for 24 h. Then, the cells were washed and fixed for 30 min in 2.5% glutaraldehyde. The samples were treated with 1.5% osmium tetroxide, dehydrated with acetone and embedded in Durcupan resin. Thin sections were stained with lead citrate and examined by TECNAI 10 electron microscopy (Philips, Eindhoven, The Netherlands) at 60 kV.

For ATG6 (Beclin-1) interference, two siRNA oligonucleotides targeting ATG6 were synthesized by GenePharma (Shanghai, China). The sequences of the sense strands of the RNAs targeting ATG6 used in this study were as follows: ATG6 siRNA-1 (si1): 5′-GUGAGAAGCAAGCCCU UAUTT-3′, ATG6 siRNA-2 (si2): 5′-CUCCAGUGCUAAGCUACAU-TT-3′. A non-specific oligo that is not complementary to any human genes was used as a negative control. The mixture of si1 and si2 was used to increase the inhibitory activity. Cells were transfected with siRNA using HiPerFect (Qiagen) according to the manufacturer’s protocol.

The pcDNA3-AKT-T7 plasmid was a gift from William Sellers (Addgene plasmid 9003). Cells were seeded into 6-well plates the day before transfection. Attractene (Qiagen) was used for transfection according to the manufacturer’s protocol. Ater 24 h of incubation, the cells were subjected to different treatments. pcDNA3 empty vectors were used as controls for transfection experiments.

Cells were seeded in 6-well culture plates at 40–50% confluence. The next day, the cells were incubated with a determined dose of Sal B. Then, the cells were trypsinized, washed twice with ice-cold PBS, and the cytosolic protein fractions were isolated using the Cell Mitochondria Isolation kit (Beyotime) according to the manufacturer’s protocol.

Western blot analysis was performed as described previously (19). Briely, proteins (40 μg) were resolved by 6–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene diluoride membranes (Millipore, Billerica, MA, USA). The membranes were then blocked in 5% (w/v) skimmed-milk for 1 h, followed by incubation with appropriate primary antibodies (diluted 1:1,000–1:5,000) overnight at 4°C. Bound antibodies were detected with peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies and the blots developed using enhanced chemiluminescence (Millipore). Western blot analysis data were quantified by ImageJ densitometric analysis and normalized by GAPDH.

All experiments were performed at least three times, and the data were presented as the mean ± standard deviation. GraphPad Prism 5.0 was used for statistical analysis. A difference was considered significant at P<0.05.

We first examined the inhibitory effect of Sal B on viability in HCC cell lines. As shown in Fig. 1A, Sal B significantly inhibited the growth of cancer cells in a dose-dependent manner, with a half maximal inhibitory concentration (IC₅₀) of 143.82 μM for SK-Hep-1 and 240.11 μM for Bel-7404 after 48 h of exposure. Colony-formation experiments also indicated that Sal B markedly inhibited the growth of HCC cells (Fig. 1B and C). We also investigated the effects of Sal B on the growth of HL-7702 cells (a normal human liver cell line). The results showed that the cytotoxic effect of Sal B on HL-7702 cells (IC₅₀ 758.63 μM) appeared much lower than that observed in SK-Hep-1 and Bel-7404 cells (Fig. 1D).

To examine the cell growth inhibition induced by Sal B related to apoptosis, Sal B-treated cells were stained with propidium iodide (PI)/Annexin V-FITC and quantified by flow cytometry. The quantification shown for apoptosis reflected the values for total apoptosis. We also determined whether apoptosis was dose- or time-dependent altered in cells treated with Sal B. Sal B induced a dose- and time-dependent increase of apoptosis in HCC cells (Fig. 2A–C).

Then we measured the mitochondrial membrane potential (ΔΨm) by flow cytometry and found that Sal B treatment led to depolarization of ΔΨm in a dose-dependent manner (Fig. 2D). Western blot analyses showed that cleaved caspase-9, cleaved caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), and cytosolic Cyto c were increased after treatment with Sal B for 24 h (Fig. 2E). The level of cleaved PARP, cleaved caspase-3, cleaved caspase-9, and cytosolic Cyto c increased 2.8-, 3.0-, 2.9- and 2.5-fold, respectively, in SK-Hep-1 cells treated with 200 μM Sal B for 24 h (Fig. 2E). The level of cleaved PARP, cleaved caspase-3, cleaved caspase-9, and cytosolic Cyto c increased 3.4-, 2.7-, 2.4- and 2.8-fold, respectively, in Bel-7404 cells treated with 200 μM Sal B for 24 h (Fig. 2E). These results demonstrated that Sal B induced apoptosis in the HCC cells.

Apoptosis and autophagy are highly interactive. To examine whether Sal B could induce autophagy, the treated cells were analyzed by western blot analysis and electron microscopy. The characteristics of autophagosomes are described as double-layer structure with cytoplasmic components. After treatment with Sal B for 24 h, most of the HCC cells displayed an extensive accumulation of double structures with a broad range of morphologies, indicating the formation of autophagosomes (Fig. 3). The significantly increased expression of LC3-II, Beclin-1 and the attenuated expression of p62/SQSTM1 were observed in cells treat with Sal B for 24 h (Fig. 4A). The level of LC3-II and Beclin-1 increased 3.1- and 2.3-fold, while the level of p62 decreased 80%, in SK-Hep-1 cells treated with 200 μM Sal B for 24 h (Fig. 4A). The level of LC3-II and Beclin-1 increased 3.5- and 2.6-fold, respectively, while the level of p62 decreased 70%, in Bel-7404 cells treated with 200 μM Sal B for 24 h (Fig. 4A).

For further confirmation, the cells were incubated with an antibody against microtubule-associated protein 1 light chain 3 (LC3). The punctate LC3 II-labeled autophagolysosome vacuoles were frequently observed in cells treated with Sal B for 48 h compared with their controls (Fig. 4B). Next, we determined whether autophagy was time-dependently altered in cells treated with Sal B. A time-dependent increase of punctate LC3-II dots was observed from 12-, 24- and 48-h in cells treated with Sal B (Fig. 4C).

The relationships between autophagy and apoptosis are complicated. There is consensus that the relationships between autophagy and apoptosis are highly depended on the tumor types and stimulus characteristic. To confirm the role of Sal B-induced autophagy in HCC cells promoted or inhibited apoptosis, we treated the cells with the autophagy inhibitor 3-MA and CQ. Addition of 5 mM 3-MA or 5 μM CQ attenuated Sal B-induced apoptosis in HCC cells (Fig. 5A). In addition, siRNAs against Beclin-1 was used to block autophagy. The level of Beclin-1 decreased 70% in SK-Hep-1 cells and 60% in Bel-7404 cells treated with siRNAs against Beclin-1 (Fig. 5B). Inhibition of autophagy attenuated Sal B-induced apoptosis in HCC cells (Fig. 5B).

Western blot analysis was used to detect the influence of 3-MA pretreatment on cleaved PARP and cleaved caspase-3 expression induced by Sal B. Autophagy level was found to significantly decrease after 3-MA pretreatment, and the expressions of cleaved PARP and cleaved caspase-3 also decreased (Fig. 5C). The level of cleaved PARP, cleaved caspase-3, and LC3-II decreased from 4.5- to 2.1-, 2.7- to 1.9- and 5.5- to 1.6-fold, respectively, in SK-Hep-1 cells treated with 200 μM Sal B and the combination of Sal B and 3-MA for 24 h (Fig. 5C). The level of cleaved PARP, cleaved caspase-3, and LC3-II decreased from 3.4- to 2.5-, 3.1- to 2.2- and 4.2-to 1.2-fold, respectively, in Bel-7404 cells treated with 200 μM Sal B and the combination of Sal B and 3-MA for 24 h (Fig. 5C). All the results demonstrated that the inhibition of autophagy in HCC cells had the potential of attenuating Sal B-induced apoptosis.

AKT/mTOR signaling pathway is the key regulatory molecule of both autophagy and apoptosis. Therefore, we investigated whether the AKT/mTOR pathway played a central role in Sal B-mediated cell death. Western blot analysis confirmed that the levels of phosphorylated AKT, mTOR and its downstream effector p70S6K and p-4EBP1 were significantly reduced by Sal B (Fig. 6A). The level of phosphorylated AKT, mTOR, p70S6K and p-4EBP1 decreased 70, 70, 70 and 60%, respectively, in SK-Hep-1 cells treated with 200 μM Sal B for 24 h (Fig. 6A). The level of phosphorylated AKT, mTOR, p70S6K and p-4EBP1 decreased 70, 80, 70 and 70%, respectively, in Bel-7404 cells treated with 200 μM Sal B for 24 h (Fig. 6A).

To further identify the role of AKT in Sal B-induced biological effects, HCC cells were transiently transfected with pcDNA3-AKT-T7 plasmid. The level of phosphorylated AKT and total AKT increased 2.0- and 5.5-fold in SK-Hep-1 cells, while 1.6- and 5.2-fold in Bel-7404 cells transfected with pcDNA3-AKT-T7 plasmid (Fig. 6D). As shown in Fig. 6B–D, enforced expression of AKT significantly attenuated Sal B-induced growth inhibition, and apoptosis. Furthermore, Sal B-induced autophagy was also decreased in the transfected cells (Figs. 6D and 7). The level of cleaved PARP and LC3-II decreased from 2.6- to 1.4- and 2.2- to 1.3-fold, respectively, in SK-Hep-1 cells treated with 200 μM Sal B and the combination of Sal B and pcDNA3-AKT (Fig. 6D). The level of cleaved PARP and LC3-II decreased from 2.7- to 1.5- and 1.8- to 1.2-fold, respectively, in Bel-7404 cells treated with 200 μM Sal B and the combination of Sal B and pcDNA3-AKT (Fig. 6D). These results suggested that overexpression of AKT can override the Sal B-induced biological effects in HCC cells. Taken together, these data demonstrate that AKT is a critical mediator in regulating Sal B-mediated biological effects.