Reagents used included fetal bovine serum (Hyclone), dimethyl sulfoxide (DMSO) (Sigma, D2650), 3-methyladenine (3-MA) (Sigma, M9281), bafilomycin A1 (Sigma, B1793), rapamycin (Sigma, R8781), Z-Val-Ala-Asp fluoromethylketone (zVAD-fmk) (Sigma, C2105), matrine (Tianyuan Biologics Plant, Xi’an, China), Annexin V-FITC apoptosis detection kit (Invitrogen, V13242), PhosSTOP Phosphatase Inhibitor Cocktail (Roche, 04906845001), and SuperSignal West Pico Chemiluminescent Substrate (Pierce, NCI5080). Antibodies were obtained from the following sources: microtubule-associated protein 1 light chain 3 (LC3) (Abcam, ab51520), Akt (Cell Signaling Technology, 9272), phospho-Akt (Ser473) (Cell Signaling Technology, 4060), mTOR (Cell Signaling Technology, 2972), phospho-mTOR (Ser2448) (Cell Signaling Technology, 2971), p70S6K (Cell Signaling Technology, 9202), phospho-p70S6K (Thr389) (Cell Signaling Technology, 9205), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Hangzhou Goodhere Biotech Co., AB-P-R 001), horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Zhongshan Goldenbridge Biotech Co., ZB-2301).

The human gastric cancer cell line SGC-7901 was obtained from the Cell Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin and 100 μg/ml of streptomycin at 37°C in a 5% CO₂ incubator. The cells in mid-log phase were used in the experiments. Matrine (purity >99%) was dissolved in sterile double distilled water at a stock concentration of 40 mg/ml, and then diluted in RPMI-1640 medium to obtain the desired concentration. zVAD-fmk and bafilomycin A1 were dissolved in DMSO and control cells were similarly treated with DMSO to a maximum final concentration of 0.2%. This concentration of DMSO did not cause any adverse morphologic response. zVAD-fmk, rapamycin and bafilomycin A1 were added 1 h before matrine treatment. 3-MA was dissolved in heated sterile double distilled water to make a 100-mM stock solution and then added to the medium for a final concentration of 5 mM. After 3 h, matrine was added for treatment.

SGC-7901 cells were seeded into tissue culture flasks, allowed to attach 24 h and then exposed to matrine with or without specific inhibitors 3-MA, zVAD-fmk, rapamycin or bafilomycin A1. After the end of specified treatment periods, cells were lysed on ice with RIPA buffer for 20 min in the presence of PhosSTOP and then centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was collected and stored in aliquots at −80°C until analysis by western blotting. Fifty micrograms protein were loaded into each well and separated by electrophoresis through 8, 10 or 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes (Millipore) using Trans-Blot Semi-Dry Cell or Mini Trans-Blot Cell apparatus (Bio-Rad). After blocking with 5% non-fat dry milk or bovine serum albumin in 1X TBST (20 mM Tris-HCl, 150 mM NaCl and 0.05% Tween-20) for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C followed by incubation with HRP-conjugated goat anti-rabbit secondary antibodies for 1 h at room temperature. The specific protein bands were developed using SuperSignal West Pico Chemiluminescent Substrate and imaged using a VersaDoc imaging system (Bio-Rad).

Cells were trypsinized with 0.25% trypsin, collected and resuspended in 100 μl of precooled phosphate-buffered saline (PBS). After adding 1 μl of propidium iodide (PI) staining solution (100 μg/ml), cells were incubated for 20 min in the dark at room temperature, supplemented with 400 μl of binding buffer and analyzed using a flow cytometer (Beckman Coulter, USA).

After 24 h of treatment, SGC-7901 cells were harvested by trypsinization, washed twice with PBS and fixed in 2.5% glutaraldehyde for 90 min at room temperature, and post-fixed in 1% osmium tetraoxide for 30 min. After washing with PBS, the cells were progressively dehydrated in ascending grades of ethanol solutions (50, 70, 95 and 100%), and embedded in Epon 812 resin. The blocks were cut into ultra-thin sections with a microtome, and were then stained with saturated uranyl acetate and lead citrate. The ultrastructure of the cells was then examined in a transmission electron microscope (JEM-1230, Jeol, Japan).

Following drug treatment, floating cells were collected and combined with adherent cells that were detached from culture dishes by treating with trypsin. Total cells were then washed with cold PBS twice and resuspended in 1X Annexin V binding buffer at a concentration of 1×10⁶ cells/ml. A single-cell suspension (100 μl) was stained with 5 μl Annexin V-FITC and 1 μl of PI for 15 min at room temperature in the dark. After the incubation period, 400 μl of 1X Annexin V binding buffer were added to each tube, followed by cytometric analysis (EPICS XL, Beckman Coulter). The extent of apoptosis was quantified as a percentage of Annexin V-positive cells, and all experiments were performed in triplicate.

To determine cell viability, the sulphorhodamine B (SRB) colorimetric assay, which estimates cell number indirectly by staining total cellular protein with the dye SRB, was used (42). SGC-7901 cells were seeded in 96-well flat bottom microtiter plates at a density of 5×10³ cells per well. Following treatment, the cells were fixed with 10% (w/v) trichloroacetic acid at 4°C for 1 h, and then stained at room temperature for 20 min with 0.4% (w/v) SRB solution. The cells were subsequently washed with 1% acetic acid five times and dissolved in 150 μl of 10 mmol/l Tris base solution (pH 10.5). The absorbance value per well at 510 nM was read using an automatic multiwell spectrophotometer (PowerWave x, Bio-Tek Instruments Inc., USA). All SRB assays were repeated three times. Cell viability was calculated according to the formula: Cell viability (%) = A510 (sample)/A510 (control) × 100.

All data are expressed as the means ± standard deviation (SD). Statistical analysis was performed using the SPSS 16.0, and p<0.05 was considered to indicate statistically significant differences. The data were analyzed by ANOVA followed by Bonferroni t-test for multiple comparisons.

We have reported that matrine induces autophagy in SGC-7901 cells which relies on the observation of cell ultrastructural changes by transmission electron microscopy and the visualization of monodansylcadaverine-labeled autophagic vacuoles by fluorescence microscopy. Here, we further examined the expression levels of LC3 which exists in cells in two forms, LC3-I and LC3-II. LC3-I residing in the cytoplasm is conjugated to phosphatidylethanolamine to form LC3-II, which is closely associated with autophagosome membranes and serves as a reliable marker to monitor autophagy. Western blot analysis of proteins from matrine-treated cells revealed the presence of two bands (Fig. 1B). Weak bands corresponding to LC3-I were found in the untreated cells whereas LC3-II was undetectable. When the cells were treated with matrine at the concentration of 1.0 mg/ml, the expression levels of LC3-II significantly enhanced in a time-dependent manner. Furthermore, LC3-II was observed as early as 3 h posttreatment with matrine. Exposure of SGC-7901 cells to matrine also resulted in a concentration-dependent increase of LC3-II protein levels at each of the treatment points indicated (Fig. 1C).

The increase in LC3-II expression could reflect either increased formation of autophagosomes due to increases in autophagic activity, or reduced turnover of autophagosomes (43). To address the issue, we assessed the influence of matrine on LC3-II levels in the presence of bafilomycin A1 or 3-MA. Bafilomycin A1 is an inhibitor of the vacuolar-type ATPase that alters the pH of acidic compartments, which blocks the fusion of autophagosomes with lysosomes, thus preventing the autophagic degradation including that of LC3-II (44). 3-MA can inhibit autophagy due to suppression of class III phosphatidylinositol 3-kinase which is essential for the initiation of the early stages of autophagy (45). The addition of 3-MA was shown to decrease LC3-II levels in matrine-treated cells, while bafilomycin A1 had their LC3-II further accumulated during matrine treatment (Fig. 1D and E). The opposite effects of 3-MA and bafilomycin A1 on LC3 are due to the two inhibitors blocking autophagy at different stages; 3-MA inhibited autophagosome formation from the beginning, whereas bafilomycin A1 prevented degradation of LC3 in autophagolysosomes and in turn increased LC3 levels (46). These findings demonstrated that the LC3-II accumulation induced by matrine was a consequence of increased autophagosome formation, indicating that matrine increased autophagic flux, rather than prevented autophagic degradation, in SGC-7901 cells.

Cell death in response to matrine was quantified by PI staining and flow cytometry, and PI positive cells were counted as dead cells. SGC-7901 cells were treated with matrine at concentrations ranging from 0 to 2.0 mg/ml for 24 h. As shown in Fig. 2A, matrine killed gastric cancer cells in a dose-dependent manner. At the concentration of 2.0 mg/ml matrine, the cell death rate reached 35.67±4.82%.

The above results showed that exposure of gastric cancer cells to matrine resulted in the extent of autophagy increasing in a dose- and time-dependent manner, which is positively correlated with the cytotoxic effect of matrine; hence, we further investigated whether matrine-induced cell death was mediated by autophagy. Furthermore, our previous study demonstrated that both apoptosis and autophagy were activated during matrine treatment (14), therefore it is necessary to further explore the interconnection between matrine-induced autophagy and apoptosis. To address these questions, we first determined whether inhibition of autophagy by 3-MA affects the cytotoxicity of matrine. The result showed that the cell death by flow cytometry in the gastric cells treated with matrine plus 3-MA sharply increased compared with matrine alone (Fig. 2B). At the same time, we used another inhibitor of autophagy at a late stage, bafilomycin A1, to further establish the role of autophagy in cell death induced by matrine. As shown in Fig. 2C, although bafilomycin A1 treatment alone had little effect on SGC-7901 cells, this agent similarly enhanced the cytotoxicity of matrine. Also, the combination of matrine with 3-MA acted cooperatively to decrease the viability of SGC-7901 cells when compared with matrine alone, measured by SRB assay (Fig. 2D). Markedly, when 3-MA inhibited matrine-induced autophagy, inspection of the cells co-treated with matrine and 3-MA using transmission electron microscopy revealed prototypical characteristics of apoptosis. As shown in Fig. 3, control cells without matrine exposure exhibited normal ultrastructural morphology. The cells treated with 1 mg/ml matrine alone showed presence of abundant autophagic vacuoles sequestrating cytoplasm and organelles and absence of chromatin changes. By contrast, after incubation with matrine in the presence of 3-MA for 24 h, the number of autophagic vacuoles decreased in the cells and some of these cells underwent apoptosis, which is characterized by cell shrinkage, plasma membrane blebbing, and chromatin condensation with margination of chromatin to the nuclear membrane. Similar effects were further confirmed by Annexin V-FITC/PI double staining that addition of 3-MA augmented matrine-induced apoptosis of gastric cancer cells compared to matrine treatment alone (Fig. 3E), indicating that matrine-induced autophagy may constitute a pro-survival compensatory mechanism, the inhibition of which drives more cells to die of apoptosis. Consistent with these observations, other studies also reported that matrine induced apoptosis in gastric cancer SGC-7901 cells (21) and MKN45 cells (20) via activation of crucial caspases such as caspase-3. Collectively, the results suggested that the cytotoxic effect of matrine on SGC-7901 cells is caused by apoptosis but not by autophagy which is a protective response by preventing cells from undergoing apoptosis, and autophagy inhibition could accelerate cell death induced by matrine.

To further explore if matrine-induced cell death is apoptotic, the cells were pretreated with a pan-caspase inhibitor zVAD-fmk for 1 h followed by exposure to matrine for 24 h. zVAD-fmk is a widely used inhibitor in characterizing apoptotic cell death as it binds irreversibly to the active site of activated caspases and inhibits caspase-mediated apoptosis (47). As shown in Fig. 4A, matrine-induced cell death was markedly reversed by zVAD-fmk, which further demonstrated apoptosis was the major form of cell death induced by matrine and may be caspase-dependent.

It has been suggested that excessive autophagy ultimately induces a type of cell death called autophagic cell death (32,48–50). Therefore, we hypothesized that if the cell death induced by matrine is indeed mediated by autophagy, the cytotoxicity of matrine is significantly enhanced after co-treatment with matrine and rapamycin which is known to be a potent autophagy inducer. As shown in Fig. 4B, however, rapamycin did not cause an additional increase of cell death quantified by flow cytometry, but instead attenuated the cell death.

The PI3K/Akt/mTOR/p70S6K signaling pathways are well-known pathways involved in the regulation of autophagy, and some anticancer agents are reported to block the pathways (37–41). Therefore, we examined the effect of matrine on the pathways, using western blotting. Akt is a downstream target of PI3K, which is phosphorylated at Tyr308 by phosphoinositide-dependent kinase 1 and at Ser473 by the mTOR complex 2 (mTORC2), resulting in its full activation (51). As shown in Fig. 5A, matrine-treated cells exhibited a dramatic increase in Akt phosphorylation at Ser473 in a dose-dependent manner. However, total Akt levels decreased in a dose-dependent manner after treatment with matrine at 3, 6 and 24 h, respectively, especially in the cells exposed to 2.0 mg/ml of matrine for 24 h. This was unexpected, as Akt activation is well known to suppress autophagy in mammalian cells. On the other hand, the increased Akt phosphorylation might, in part, limit clinical anticancer efficacy of matrine due to its suppression of apoptosis.

It has been reported that mTOR inhibitors such as rapamycin induce increased levels of phospho-Akt via negative feedback regulation of insulin receptor substrate-1 (52). As expected, rapamycin increased levels of phospho-Akt; notably, co-treatment with matrine and rapamycin was able to augment this counterproductive increase (Fig. 5B). The activation of Akt may be responsible for the finding that addition of rapamycin contributed to the decrease of the cell death induced by matrine (Fig. 4B).

The serine/threonine kinase mTOR is a major downstream substrate of Akt and is at the core of two distinct multiprotein kinase complexes, mTOR complex 1 (mTORC1) and mTORC2. mTORC1 activation by the PI3K/Akt pathway results in phosphorylation and activation of p70S6K at Thr389 and mTORC2 has a role in activating Akt through Ser473 phosphorylation (53). As shown in Fig. 6, matrine slightly increased phosphorylation of mTOR at Ser2448 in gastric cancer cells at 6 h, which was accompanied by a slight decrease in total mTOR protein in a dose-dependent manner. We also observed matrine treatment for 6 h modestly increased p70S6K at Thr389 phosphorylation and without any effect on the total p70S6K level. These results were counterproductive, as activated mTOR is generally considered to be involved in the negative control of mammalian autophagy.