Oleanolic acid induces autophagic death in human gastric cancer cells in vitro and in vivo

Chemicals and reagents

OA, 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 3-methyladenine (3-MA) were purchased from Sigma. Antibodies specific for LC3B, Beclin-1, mTOR, phospho-mTOR, AKT, phospho-AKT, AMPK (CST, 5831), phospho-AMPK, ERK1/2, phospho-ERK1/2, PTEN, phospho-PTEN, phospho-ULK1, phospho-PI3 K, phospho-p38, and β-actin were purchased from Cell Signalling Technology. A secondary antibody conjugated with Alexa Fluor680 was purchased from Jackson ImmunoResearch Laboratories. All other chemicals were of the highest purity available.

Cell lines and cell culture

The human normal gastricepithelial cell line GES-1 and RGM-1, human gastric cancer cell lines SGC-7901, MGC-803 and BGC-823 obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All cell lines were routinely cultured in RPMI 1640 medium (Gibco), which contained 10% fetal calf serum (Gibco), 100 U/mL penicillin, and 100 U/mL streptomycin, in a humidified cell incubator at 37°C with an atmosphere of 5% CO2.

Cell viability assay

Cell viability was measured by the MTT method. Briefly, exponentially growing cells were seeded in 96-well plates, then incubated with at various concentrations of OA for 24, 48, or 72 h. MTT was then added to each well, and the cells were incubated for a further 4 h at 37°C. The formazan precipitate was dissolved in 150 µL DMSO, and the absorbance at 490 nm was measured using a microplate reader (Thermo). Each test was repeated at least three times.

Western blot analysis

Approximately 30 µg of lysed protein was separated by 10–15% sodium dodecyl sulphate PAGE, transferred to a nitrocellulose blot membrane (Bio-Rad), and then blocked for 1 h in blocking buffer (5% bovine serum albumin solution and 0.1% Tween 20 in Tris-buffered saline (TBST)). The membrane was incubated with the primary antibody overnight at 4°C. After three washes, the blots were subjected to a secondary antibody for 1 h in blocking buffer. Proteins were visualised using an Odyssey Imager (LI-COR).

GFP-RFP-LC3transfection and fluorescence

The cells were transfected with the plasmid GFP-RFP-LC3 using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. At 48 h after transfection, cells were treated with OA for 24 h. The fluorescence of GFP-RFP-LC3 was observed; autophagosomes (yellow dots) and autolysosomes (free red dots) in each cell were counted under an Olympus FV1000 confocal microscope. The GFP-RFP-LC3 plasmid was kindly provided by Prof. Mao Xiang (Sun et al., 2012).

Transmission electron microscopy (TEM)

The collected cells were fixed overnight in 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH7.0). After three washes, the specimens were fixed with 1% OsO4 for 1 h. The specimen was dehydrated using a graded series of ethanol (30, 50, 70, 80, 90 and 100%) for 15 min at each step and then transferred to pure acetone for 20 min. The specimen was placed in a 1:1 mixture of pure acetone and final resin mixture for 1 h, transferred to a 1:3 mixture of pure acetone and final resin mixture for 3 h, and then to the final spur resin mixture overnight. Specimens were heated at 37°C for more than 9 h. Finally, the specimens were sectioned with a LEICA EM UC7 ultramicrotome. Sections were stained with uranyl acetate and alkaline lead citrate for 5 min and observed with a Hitachi Model H-7650 TEM.

Transient transfection with small interfering siRNA

siRNA targeting Beclin-1 and CypD were purchased from Santa Cruz. Cells were transfected with the corresponding siRNA of the target gene or scrambled control siRNA by Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer's instructions. Knockdown efficiency was assessed by Western blot after 48 h of transfection.

Xenograft model in nude mice

The procedure of establishing MGC-803 xenografts in nude mice was approved by the Zhejiang University Laboratory Animal Centre (Hangzhou, China). Briefly, 4 × 10⁶ MGC-803 cells were subcutaneously injected into the right flanks of 6-week-old female nude BALB/c mice, which were then randomly divided into three groups (n = 8). Beginning at day 7, the MGC-803 + OA group was orally treated with OA at 100 mg/kg body weight every day. The MGC-803 + OA + 3-MA group was intraperitoneally injected with 10 mM 3-MA in 50 µL of DMSO at 3-day intervals and received oral OA treatment as well. The MGC-803 group was injected with 50 µL of DMSO as acontrol. Tumour diameters were measured with a calliper at 3-day intervals beginning at day 7. Tumour volume = (shortest diameter²× longest diameter)/2. All of the mice were sacrificed on day 37, and tumours were harvested.

Statistical analysis

Data are presented as the means ± standard deviations (SD). Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A value of P < 0.05 was considered statistically significant.

OA inhibits the growth of human gastric cancer SGC-7901, MGC-803 and BGC-823 cells

To identify the potential of OA for treating human gastric cancer, GES-1, RGM-1, SGC-7901, MGC-803 and BGC-823 cells were treated with OA, and cell viability was determined by the MTT assay. We found that OA inhibited the growth of SGC-7901, MGC-803 and BGC-823 cells in a dose- and time-dependent manner (Figures 1D and 1F). The 50% inhibitory concentrations (IC50) for SGC-7901 cells were 39.5, 25.9 and 21.2 µM after treatment with OA for 24, 48 and 72 h, respectively. The IC50 values in MGC-80-3 cells and BGC-823 cells after treatment with OA were 30.6 and 39.4 µM (for 24 h), 24.0 and 30.1 µM (for 48 h), 20.4 and 22.4 µM (for 72 h), respectively. In contrast, only a small percentage of inhibition was detected in OA-treated GES-1 and RGM-1 cells (Figures 1B and 1C).

OA induces autophagy in human gastric cancer SGC-7901, MGC-803 and BGC-823 cells

Beclin-1 controls the initiation of autophagocytosis and activates microtubule-associated Protein 1; LC3, whose cytosolic form (LC3-I) is lipidated to the LC3-II form during autophagy, is a specific marker for autophagosome formation. As shown in Figure 2A, compared with the untreated group, OA induced a noticeably greater amount of Beclin-1 and LC3-II in SGC-7901, MGC-803 and BGC-823 cells after OA treatment for 24 h.

To confirm the characteristics of autophagy induced by OA, the accumulation of GFP-RFP-LC3 puncta, autophagosomes and autolysosomes in SGC-7901, MGC-803 and BGC-823 cells was investigated by observation with transmission electron microscopy (TEM) and confocal microscopy. OA-treated SGC-7901, MGC-803 and BGC-823 cells showed cytoplasmic accumulation of autophagosomes, a morphological marker of autophagy as determined by TEM, after OA treatment for 24 h (Figure 2B). In addition, confocal microscopy analysis revealed that, in contrast to the relatively homogeneous distribution in control cells, GFP-RFP-LC3 puncta accumulated in the cytoplasm of OA-treated cells (Figures 2C–2E). Autophagosomes (yellow dots) and autolysosomes (free red dots) in each cell were counted as shown in Figure 2F. Pretreatment with 2 mM 3-methyladenine (3-MA, an autophagy inhibitor) for 2 h obviously suppressed the accumulation of autophagosomes and autolysosomes (Figures 2C–2F). These data indicate that autophagy is induced by OA in SGC-7901, MGC-803 and BGC-823 cells.

OA induces the inhibition of mTOR

mTOR has been reported to be involved in the cell autophagy process, and the suppression of mTOR phosphorylation is considered to be responsible for the early triggering of autophagy, which is regulated by the PI3 K/AKT, ERK/MAPK and AMPK signalling pathways (Dunlop and Tee, 2014; Shimobayashi and Hall, 2014). We investigated the role of mTOR in OA-induced autophagy. As shown in Figure 3, OA treatment for 24 h decreased the phosphorylation of PI3 K, AKT, ERK1/2, p38 and mTOR, but increased the phosphorylation of AMPK. ULK1, a kinase essential for autophagy initiation, was controlled by phospho-mTOR, OA treatment increased phosphorylation of ULK1 indicated an increasing level of autophagic initiation. These data suggest that OA induces autophagy by suppressing phospho-mTOR through inhibition of the PI3 K/AKT and ERK/p38 MAPK signalling pathways and activation of the AMPK signalling pathway.

Inhibition of autophagy attenuates OA-induced cytotoxicity

Because Beclin-1 controls the initiation of autophagocytosis, suppressing Beclin-1 expression may attenuate OA-induced cytotoxicity and autophagy. We found that knocking down the expression of Beclin-1 in SGC-7901, MGC-803 or BGC-823 cells by siRNA led to a reduction in OA-induced cell death, autophagosomes and autolysosomes (Figures 4A, 4B and 4D). To further examine whether the OA-induced cytotoxicity of SGC-7901, MGC-803 and BGC-823 cells was caused by autophagy, we examine the suppression of OA-induced cytotoxicity and autophagy via the autophagy inhibitor 3-MA. As illustrated in Figures 4C and 4E, a 2 h pretreatment with 2 mM 3-MA significantly reduced the OA-induced cytotoxicity and accumulation of GFP-RFP-LC3 vacuoles in SGC-7901, MGC-803 and BGC-823 cells. Taken together, our data indicate that OA-induced cytotoxicity in SGC-7901, MGC-803 and BGC-823 cells is caused by autophagy.

Growth of human gastric cancer MGC-803 cells in nude mice is inhibited by OA treatment

We applied the tumour xenograt mouse model of MGC-803 cells to investigate the therapeutic potential of OA in gastric cancer in vivo. As shown in Figures 5A and 5B, in the drug-treated mouse group, OA decreased the growth of MGC-803 tumours and delayed the occurrence of tumour formation compared with the control. 3-MA treatment abolished the inhibitory effect of OA on tumour growth. After sacrificing the mice and observing tumour formation, we confirmed that OA-induced autophagy was decreased in 3-MA-treated MGC-803-originating tumours by examining the expression of LC3-II and Beclin-1 (Figure 5C).