β-Elemene-induced autophagy protects human gastric cancer cells from undergoing apoptosis

The human gastric cancer cells MGC803 and SGC7901 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium (Gibco) containing 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 mg/mL) at 37°C under an atmosphere of 95% air and 5% CO2. The cells were routinely subcultured every 2-3 days, and were all from the logarithmic phase of growth.

β-Elemene was obtained from Yuanda Pharmaceuticals (Dalian, China). 3-Methyladenine (3-MA) and chlorochine (CQ) were purchased from Sigma-Aldrich (St. Louis, Mo. USA). LysoTracker and Hoechst33342 were from Invitrogen (USA). Anti-Bcl-2, anti-Bax, anti-Survivin, anti-actin and anti-Akt antibodies were purchased from Santa Cruz Biotechnology (USA). Anti-LC3, anti-Beclin 1, anti-Atg5, anti-Atg9 and anti-Atg16L antibodies were from Novus Biological (Littleton, CO, USA). Anti-caspase 3, anti-poly(ADP-ribose) polymerase (PARP), anti-phospho-Akt (Ser-473), anti-phospho-mTOR, anti-mTOR, anti-phospho-p70S6K1, and anti-p70S6K1 antibodies were purchased from Cell Signaling Technology (USA).

Cell viability was measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cells were seeded at 5 × 10⁴ cells/well in 96-well plates, incubated overnight and then exposed to the indicated concentrations of β-elemene for the indicated times. Thereafter, 20 μl of MTT solution (5 mg/mL) was added to each well, and the cells were incubated for another 4 h at 37°C. After removal of the culture medium, the cells were lysed in 200 μl of dimethylsulfoxide (DMSO), and the optical density (OD) was measured at 570 nm with a microplate reader (Model 550; Bio-Rad Laboratories, USA). The following formula was used: cell viability = (OD of the experimental sample/OD of the control group) × 100%.

Cells were seeded at 3 × 10⁵ cells/well in 6-well plates, incubated overnight and then exposed to the indicated concentrations of β-elemene for the indicated times. Cells were collected and incubated with 1 μg/mL Annexin V for 20 min in the dark. Finally, the samples were evaluated by flow cytometry and the data were analyzed using WinMDI software.

For the analysis of green fluorescent protein-fused LC3 (GFP -LC3) localization, MGC 803 cells were transfected with a plasmid encoding GFP-LC3 (kindly provided by Høyer-Hansen M, Danish Cancer Society) and stably expressing cells were selected with changes of media containing 200 μg/mL of G418. Transfection was performed using Lipofectamine 2000 reagent (Invitrogen, USA), according to the manufacturer's instructions. After treatment of 10 or 50 μg/mL β-elemene for 24 h, cells were incubated with 50 nmol/L LysoTracker for 30 min at room temperature, and the nucleus was stained with Hoechst33342. The images were obtained with a fluorescence microscope (Olympus, Japan). The detection of punctated GFP-LC3 co-locolized with LysoTracker indicated the formation of autophagosomes.

Cells were treated and collected by trypsinization, then fixed with 2.5% phosphate-buffered gluteraldehyde, postfixed in 1% phosphate-buffered osmium tetroxide. Cells were then embedded, sectioned, double stained with uranyl acetate and lead citrate, and analyzed using a JEM-1200EX transmission electron microscope (JEOL, Japan).

Cells were washed twice with ice-cold PBS and solubilized in 1% Triton lysis buffer [1% Triton X-100, 50 mmol/L Tris-Cl (pH 7.4), 150 mmol/L NaCl, 10 mmol/L EDTA, 100 mmol/L NaF, 1 mmol/L Na₃VO₄, 1 mmol/L PMSF and 2 μg/mL aprotinin] on ice, then quantified using the Lowry method. Cell lysate proteins (50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes (Immoblin-P; Millipore, USA). The membranes were blocked with 5% skim milk in TBST buffer [10 mmol/L Tris (pH 7.4), 150 mmol/L NaCl and 0.1% Tween-20] at room temperature for 1 h and incubated overnight at 4°C with the indicated primary antibodies. After the membranes were washed with TBST buffer, they were reacted with the appropriate horseradish peroxidase-conjugated secondary antibodies for 30 min at room temperature. After extensive washing with TBST buffer, the proteins were visualized with enhanced chemiluminescence reagent (SuperSignal Western Pico Chemiluminescent Substrate; Pierce, Rockford, IL, USA). The images were analyzed using NIH Image J software.

Cells were seeded at 5 × 10⁴cells/well in 12-well plates and treated with either 50 μg/mL β-elemene or 20 μmol/L CQ, or co-treatment with β-elemene and CQ for 6 h. Cells were then plated (in triplicate) at 200 cells per well into 12-well plates with fresh drug-free medium. Cells were incubated for an additional 14 days, and the clones in each well were stained, counted and photographed.

Small interfering RNA corresponding to the human Beclin 1 cDNA sequence (5'-CAGTTTGGCACAATCAATAtt-3') [19] and a control siRNA (5'-UUCUCCGAACGUGUCACGUtt-3') were from Genechem Co. (Shanghai, China). Cells were transfected with either Beclin 1 siRNA or control siRNA at 100 nmol/L using DharmaFECT transfection agent (Dharmacon Research, CO, USA) according to the manufacturer's guidelines. Forty-eight hours after transfection, the cells were subcultured for further use. The expression of Beclin 1 was verified by western blotting.

The experiments were repeated at least three times. Data are expressed as the means ± SD. Differences in the results for two groups were evaluated by the Student's t-test. P < 0.05 was considered to be statistically significant.

Human gastric cancer MGC803 cells were treated with β-elemene at concentrations ranging from 10 μg/mL to 200 μg/mL for 24, 48 or 72 h. Cell viability assays showed that β-elemene inhibited cell growth in a dose-dependent manner (Figure 1A). The IC₅₀ values of β-elemene at 24, 48 and 72 h were 80.03 μg/mL, 56.03 μg/mL and 45.05 μg/mL, respectively. Flow cytometry assays showed a significant increase in the apoptotic population among the cells treated with β-elemene at 24 h (Figure 1B). To further confirm that apoptosis was induced by β-elemene, western blotting was performed to detect the cleavage of caspase 3 as well as PARP. As shown in Figure 1C β-elemene clearly cleaved pro-caspase 3 and PARP to their active forms. The effects of β-elemene on the expression of apoptosis associated proteins were further investigated. Western blotting showed that β-elemene had little effect on the expression of Bax or Bcl-2, but significantly down-regulated the level of Survivin (Figure 1D and 1E). These data indicated that β-elemene inhibits cell viability through inducing apoptosis in human gastric cancer cells.

It has been reported that some lipid-soluble anti-tumor agents such as oleandrin could simultaneously induce apoptosis and autophagy [20], so we asked if there was any autophagy in the cells treated with β-elemene. MGC803 cells stably expressing GFP-LC3 were treated with 10 or 50 μg/mL β-elemene for 24 h, and the localization of GFP-LC3 was evaluated under fluorescent microscopy. As shown in Figure 2A, only a few LC3-positive puncta were observed in untreated control cells. However, in the cells treated with 10 μg/mL β-elemene, over 30% of cells were observed with LC3-positive puncta, and in the cells treated with 50 μg/mL β-elemene, more than 90% of cells showed LC3-positive puncta. GFP-LC3 co-localized with LysoTracker after treatment with β-elemene, which suggests the accumulation of autophagosomes. The formation of autophagosomes was further confirmed by transmission electron microscopy. Upon treatment of 50 μg/mL β-elemene many autophagic vesicles, double membrane enclosed vesicle containing engulfed organelles, were observed in the cytoplasm (Figure 2B). Meanwhile, β-elemene treatment also significantly increased LC3-II levels as demonstrated by western blotting (Figure 2C). These data indicate that β-elemene treatment not only results in apoptosis, but also induces autophagy.

Autophagy is regulated by a group of proteins called Atg (autophagy-related) proteins [10]. Some stimuli can modulate autophagy simply by changing the expression of certain Atg protein [21]. To determine if β-elemene could affect the expression of Atg proteins, MGC803 cells were treated with 50 μg/mL β-elemene for 24 h and the levels of some Atg proteins were detected by western blotting. After treatment with β-elemene, the level of Atg5-Atg12 conjugated protein was up-regulated, whereas the expression of Beclin1, Atg5, Atg9 and Atg16L was not affected significantly (Figure 3A and 3B). These data suggest that the up-regulation of Atg5-Atg12 conjugated protein might contribute to the induction of autophagy by β-elemene.

It has been well documented that the PI3K/Akt/mTOR/p70S6K1 signalling pathway plays a key role in regulating both apoptosis and autophagy, so the effects of β-elemene on the activity of this pathway were studied next. Cells were treated with 10 or 50 μg/mL β-elemene for 24 h, and the levels of phospho-Akt, phospho-mTOR and phospho-p70S6K1 were detected by western blotting. After treatment with 50 μg/mL β-elemene, the level of phospho-Akt was obviously down-regulated, leading to the down-regulation of downstream phospho-mTOR as well as phospho-p70S6K1 (Figure 4A). A low dose (10 μg/mL) of β-elemene had little effect on PI3K/Akt/mTOR/p70S6K1 activity. Interestingly, a transient activation of PI3K/Akt/mTOR/p70S6K1 was observed after the cells were exposed to β-elemene for short times. As shown in Figure 4B, the level of phospho-Akt increased after 4 h, stayed active until 8 h, and went down after 16 h. Similar changes were observed on the phosphorylation of mTOR and p70S6K1. The cleavage of PARP and conversion of LC3 I to LC3 II was also seen following treatment with 50 μg/mL β-elemene at 16 h and 24 h, consistent with the change of PI3K/Akt/mTOR/p70S6K1 activity (Figure 4C). These data indicated that β-elemene induced apoptosis and autophagy might be due to its inhibition of the PI3K/Akt/mTOR/p70S6K1 signalling pathway.

Since autophagy can result in both survival and cell death, we then asked whether β-elemene-induced autophagy is protective or pro-apoptotic. MGC803 cells were treated with either 50 μg/mL β-elemene or 20 μmol/L CQ (an inhibitor of autophagy which blocks the fusion of autophagosomes with lysosomes, stopping autophagy at the late phase), or co-treated with β-elemene and CQ for 24 h. Cell viability assays showed that co-treatment with β-elemene and CQ significantly decreased cell viability, compared with the cells treated with β-elemene alone (48.50 ± 5.07% vs. 78.27 ± 1.57%, P<0.05) (Figure 5A). Co-treatment with β-elemene and CQ also significantly reduced the clone formation ability of the cells and increased the apoptotic population compared with the cells treated with β-elemene alone (Figure 5B and 5C). To confirm the effect of autophagy inhibition by the pharmacologic agent CQ on β-elemene-induced apoptosis, an RNA interference approach was used to knock down the expression of Beclin 1. Figure 5D shows that the level of Beclin 1 was significantly decreased in Beclin 1 siRNA-treated cells. Compared with the results in siRNA controls, knockdown of Beclin 1 decreased significantly the cell viability, and enhanced β-elemene-induced apoptosis (Figure 5 E and 5F). These data indicate that blockage of autophagy enhanced the antitumor effect of β-elemene in MGC803 cells.

To prove that the apoptosis and autophagy induced by β-elemene is not cell-specific, we examined the antitumor effect of β-elemene on another human gastric cancer cell line, SGC7901. We found that β-elemene inhibited the viability of SGC7901 cells in a dose-dependent manner, and the IC₅₀ values at 24, 48 and 72 h were 89.68 μg/mL, 75.88 μg/mL and 67.13 μg/mL, respectively (Figure 6A). β-Elemene inhibited mTOR activity and induced apoptosis and autophagy, which were evidenced by the cleavage of PARP and the conversion of LC3-I to LC3-II (Figure 6B). The contribution of autophagy to β-elemene-induced apoptosis in SGC7901 cells was evaluated further by co-treating the cells with β-elemene and the autophagy inhibitor, 3-MA or CQ. Compared with the cells treated with β-elemene alone, co-treatment with β-elemene and 3-MA or CQ reduced significantly the viability and clone formation ability of the cells, and increased the apoptotic population (Figure 6C, D and 6E). Similar results from these two human gastric cancer lines indicate that autophagy induced by β-elemene served in a protective manner, and blockage of autophagy enhanced the anti-tumor effect of β-elemene in human gastric cancer cells.