TMPZ was acquired from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). TMPZ was dissolved in distilled water to produce a stock solution of 1 M concentration. RPMI-1640 medium was purchased from Gibco-BRL (Carlsbad, CA, USA). Fetal calf serum (FCS) was obtained from HyClone (Logan, UT, USA). Compound C was purchased from Merck KGaA (Darmstadt, Germany). N-acetylcysteine (NAC), trypsin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), Triton X-100, HEPES, NP-40, phenylmethylsulfonyl fluoride (PMSF), aprotinin and leupeptin were obtained from Sigma (St. Louis, MO, USA). The anti-phospho-AMPK (Thr¹⁷²), anti-AMPKα, anti-cytochrome c and anti-Bax antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The anti-caspase-3 and -9 and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All the reagents used in the study were of the highest purity.

The SGC7901 human gastric adenocarcinoma cell line was obtained from the Chinese Academy of Medical Sciences Cell Center of Basic Medicine (Beijing, China). The cells were maintained in RPMI-1640 medium containing 10% FCS at 37°C, in a humidified incubator with a mixture of 95% air and 5% CO₂.

Cell viability was analyzed using the MTT assay. Briefly, SGC7901 cells were seeded at 1×10⁴ cells/well in flat-bottomed 96-well plates. At 24 h following TMPZ treatment, 100 μl MTT was added to each well and the cells were incubated at 37°C for 4 h, resulting in the formation of MTT formazan crystals. Following incubation, the supernatants were removed and the formazan crystals were solubilized in dimethylsulfoxide. The plates were thoroughly shaken and the absorbance of each well was measured at 570 nm using a Bio-Tek microplate reader (Bio-Rad, Hercules, CA, USA).

Apoptosis was identified by flow cytometry with an Annexin V-FITC Apoptosis Detection kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. The cells were washed twice with cold phosphate-buffered saline (PBS) and were then resuspended in a buffer solution at a final concentration of 1×10⁶ cells/ml. Annexin V-FITC (5 μl) and PI (5 μl) were added to the cells, which were resuspended in 500 μl of 1X binding buffer. The cells were gently vortexed and incubated in the dark at room temperature for 15 min. The cells were then analyzed using a FACSAria™ flow cytometer (BD Biosciences) within 1 h. A minimum of 10,000 cells was analyzed in each treatment group and the analysis of apoptotic cells was performed using CellQuest software (BD Biosciences).

The cells were rinsed twice with ice-cold PBS and then incubated for 30 min in lysis buffer (50 mM Tris-HCl, pH 8.0; 1% sodium deoxycholate; 1% NP-40; 0.1% SDS; 1 mM PMSF; 10 μg/ml aprotinin and 10 μg/ml leupeptin). The cells were then scraped and centrifuged at 12,000 × g for 10 min at 4°C. Extracts of the cytoplasm and mitochondria were prepared using the Mitochondria/Cytosol Isolation kit (Applygen Technologies, Beijing, China) according to the manufacturer’s instructions. The protein concentration in the lysates was quantified using a BCA Protein Assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Cell lysates and fractionated extracts were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dried milk in 10 mM Tris (pH 7.5), 100 mM NaCl and 0.1% Tween-20 for 2 h at room temperature. The proteins were probed with antibodies against the target proteins and the protein bands were visualized by exposure to X-ray film. The bands were quantified by scanning the film on a GDS-8000 UVP photo scanner (Bio-Rad). The monoclonal anti-β-actin antibody was used as an internal control for loading.

The levels of ROS were measured using a Reactive Oxygen Species Assay kit (Applygen Technologies). Briefly, the intracellular formation of ROS in the SGC7901 cells was determined using 2′7′-dichlorodihydrofluorescein diacetate (DCFH-DA) molecular probes. DCFH-DA is a membrane-permeable indicator of ROS levels, and is non-fluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell. Thus, a reaction between these probes and intracellular ROS yields the fluorescent molecule DCF, which may be used as a measure of intracellular ROS levels. Briefly, SGC7901 cells were harvested, washed with PBS and loaded with 10 μM DCFH-DA in serum-free DMEM in the dark for 30 min at 37°C. The excess dye was flushed off and the cells were resuspended in serum-free DMEM. Fluorescence intensity was quantified using a BD FACSVantage™ SE (BD Biosciences).

The ΔΨm was evaluated using a Mitochondrial Membrane Potential Assay kit with JC-1 (Beyotime Biotechnology Co., Jiangsu, China). JC-1 is a mitochondria-specific probe and a fluorescent cationic dye that exhibits potential-dependent accumulation in mitochondria by a fluorescence emission shift from green (530 nm) to red (590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red-green fluorescence intensity ratio. The cells were plated at a density of 5×10⁵ cells/well in 6-well plates. Following treatment, the cells were harvested, washed with PBS and incubated with culture medium (without FBS) containing JC-1 probes (2 mM) for 30 min at 37°C in the dark. Following the removal of the JC-1 probes, the cells were washed with PBS, harvested by trypsinization and resuspended in PBS. The amount of JC-1 retained by 10,000 cells per sample was measured at 530 nm (green fluorescence) and 590 nm (red fluorescence) with a flow cytometer and analyzed with CellQuest Alias software (BD Biosciences). CCCP-treated samples were used to perform standard compensation analysis. Data are presented as the ratio of red to green signals (590/530 nm).

Each value is expressed as the mean ± standard deviation from at least three independent experiments. The differences in the means between groups were tested by one-way analysis of variance followed by the Student-Newman-Keuls test (comparisons among multiple groups). P<0.05 was considered to indicate a statistically significant difference.

To investigate the antitumor effect of TMPZ on SGC7901 gastric cancer cells in vitro, cell viability and apoptosis were assessed. Following the administration of TMPZ (0–8 mM for 24 h) to SGC7901 cells, a contrasting effect of TMPZ towards tumorigenic cells was observed. Treatment with TMPZ at the higher doses of 4, 6 and 8 mM for 24 h resulted in reduced cell viabilities of 67.8±5.4, 42.6±4.5 and 22.5±3.5% in SGC7901 cells (P<0.01), respectively, whereas treatment with lower doses of TMPZ (0.5, 1 and 2 mM) demonstrated no effect on cell viability (Fig. 1). Furthermore, the analysis of apoptosis was concordant with the results observed in the cell viability experiments. The percentages of apoptotic cells were 16.7±2.1, 25.8±3.7 and 42.3±5.2% in SGC7901 cells treated with the respective doses of TMPZ (4, 6 and 8 mM) for 24 h (Fig. 2). These findings confirmed that a high dose of TMPZ confers significant cytotoxicity, which may be beneficial for anticancer treatment. Therefore, the higher doses of TMPZ (4, 6 and 8 mM) were selected for further experimentation.

A number of natural compounds for the treatment of cancer have been demonstrated to cause increased levels of cellular ROS generation (9,10). To determine whether this mechanism is responsible for the antitumor effect of TMPZ, the generation of ROS was investigated. The results demonstrated that TMPZ treatment significantly elevated the ROS levels in a dose-dependent manner in SGC7901 cells. These data were further confirmed by incubating the cells with NAC, which is a scavenger of ROS (11). Treatment with NAC almost completely abrogated the release of ROS in SGC7901 cells and inhibited TMPZ-induced cytotoxicity (Fig. 3).

Numerous studies have implicated AMPK in cancer growth and metabolism (?). It has previously been suggested that stimulating the release of ROS may lead to the activation of AMPK (12); therefore, we investigated whether AMPK activation may result from TMPZ treatment. The results from this study revealed that high doses of TMPZ significantly upregulated the phosphorylation of AMPK in SGC7901 cells in a dose-dependent manner. To determine whether the activation of AMPK is involved in the accumulation of ROS, the cells were incubated with TMPZ in the presence of 5 mM NAC for 24 h. NAC almost completely inhibited TMPZ-induced AMPK phosphorylation, demonstrating that AMPK was activated by TMPZ-induced ROS accumulation (Fig. 4).

To verify whether the AMPK signaling pathway was responsible for the antitumor effect of TMPZ that was observed in SGC7901 cells, compound C, an inhibitor of AMPK was used (13). Pretreatment with 10 μM compound C for 15 min inhibited TMPZ-induced apoptosis (Fig. 5A). We also examined the mechanism by which AMPK mediated TMPZ-induced apoptosis. Compound C did not suppress TMPZ-induced ROS accumulation (Fig. 5B), indicating that compound C does not inhibit TMPZ-induced cell death by suppressing ROS accumulation. It is well-known that the majority of intrinsic death signals converge onto the translocation of Bax, a pro-apoptotic member of the Bcl-2 family, from the cytosol to the mitochondria (14). Our results demonstrated that TMPZ induced the translocation of Bax from the cytosol to the mitochondria and that this translocation was significantly inhibited by compound C. In addition, compound C significantly inhibited TMPZ-induced mPTP opening and depolarization of the mitochondria. The JC-1 red/green fluorescence intensity ratios were 23.4±1.5 and 82.7±4.7% in SGC7901 cells treated with 8 mM TMPZ alone and 8 mM TMPZ combined with compound C pretreatment, respectively (Fig. 5C). It is well-known that the majority of intrinsic death signals converge onto the translocation of Bax, a pro-apoptotic member of the Bcl-2 family, from the cytosol to the mitochondria (14). Our results demonstrated that TMPZ induced the translocation of Bax from the cytosol to the mitochondria and that this translocation was significantly inhibited by compound C. Furthermore, compound C significantly inhibited the TMPZ-induced release of cytochrome c and the activation of caspase-9 and -3 (Fig. 5D). Thus, ROS-induced AMPK activation is important in initiating the mitochondria-mediated apoptosis of SGC7901 cells treated with TMPZ.