Human gastric carcinoma AGS cells were purchased from the American Type Culture collection (Manassas, VA, USA). The cells were cultured at 37°C in a 5% CO₂ atmosphere with Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Capsaicin was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and prepared as an ethanol stock solution, which was then diluted in intracellular solution prior to use.

Cell viability was determined using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay. Briefly, 5×10⁵ cells/well were seeded in 96-well plates and allowed to adhere overnight. The concentrations of capsaicin used in this experiment were 0, 10, 20, 30, 60, 80, 100, 200 and 300 μM. Each group contained three wells. After the samples were treated with capsaicin for 12 h, 10 μl of MTT (0.5 mg/ml; Sigma) was added to each well, and the cells were incubated at 37°C for 4 h. The reaction was then stopped by lysing the cells with 200 μl of dimethyl sulfoxide (DMSO) for 15 min. Quantification measurements (optical density) were obtained at a wavelength of 450 nm using spectrophotometric analysis.

To evaluate the impact of capsaicin on apoptosis, FACS analysis was performed. Cell apoptosis was assessed using the Annexin V assay (Annexin V-FITC apoptosis detection kit; BD Pharmingen, San Jose, CA, USA). AGS cells (1×10⁵ cells) were treated with various concentrations (0–300 μM) of capsaicin for 12 h, respectively. Annexin V-FITC and propidium iodide (PI) labeling was then performed according to the manufacturer’s instructions. The cells were double-stained with Annexin V-fluorescein isothiocyanate (FITC) and PI, followed by flow cytometry. To quantify early apoptotic cells, flow cytometry was used to assess binding of fluorescence-labeled Annexin V to externalized phosphatidylserine. PI uptake was measured to assess cells in the late stages of apoptosis or cells that sustained direct plasma membrane damage.

Total protein was lysed with RIFA buffer (1 M Tris-HCl, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA) with 1 mM phenylmethanesulfonyl fluoride (PMSF) and Halt™ protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). Protein concentrations of the cell lysate were quantified using BCA™ protein assay (Thermo Scientific) with bovine serum albumin (BSA) as a standard. Total protein (10–20 μg) was subjected to electrophoresis on 10% SDS-polyacrylamide gels and then transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 h in blocking solution (5% BSA in TBS-Tween 20 buffer) and sequentially blotted with primary antibodies at 4°C overnight. Antibodies against c-Jun N-terminal kinase (JNK), phospho-JNK, extracellular signal-regulated kinase 1/2 (ERK 1/2), phospho-ERK 1/2 (1:1,000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA), p38 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK, caspase 3, cleaved caspase 3, Bcl-2, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). After rinsing in TST, the membrane was incubated with horseradish peroxidase (HRP)-labeled anti-rabbit or anti-mouse immunoglobulins as the secondary antibody (1:5,000 dilution, Santa Cruz Biotechnology, Inc., Santa Cruz, USA) at room temperature for 1 h. Enhanced chemiluminescence was used to visualize the bands by LAS 3000 (Fuji Film, Tokyo, Japan).

The Student’s t-test was used to determine the significance of differences between treatments and respective controls. Values are expressed as means ± SE from at least three independent experiments. P<0.05 was considered to indicate statistical significance. Statistical analysis was assessed using Statistical Package for the Social Sciences (SPSS/PC 20.0, Chicago, IL, USA).

An MTT assay was used to assess the potential effects of capsaicin on the proliferation of human gastric cancer cells that had been exposed to capsaicin for 12 h at different concentrations. After treatment with capsaicin at concentrations ranging from 10 to 300 μM for 12 h, the cell proliferation was significantly lower than that of the untreated corresponding control groups (P<0.001). Capsaicin decreased the proliferation of the gastric cancer cell line in vitro in a dose-dependent manner (Fig. 1).

Regulation of apoptosis is crucial for the preservation of homeostasis and morphogenesis of human tissue (3). Disturbance of these processes by aberrantly extending cell viability or favoring accumulation of transforming mutation is thought to contribute to carcinogenesis (3,4). To evaluate the impact of capsaicin on apoptosis, we performed FACS analysis. Fig. 2 shows flow cytometric plots obtained with Annexin V-FITC/PI assay after a 12-h exposure to different concentrations (0–300 μM). The number of early and late apoptotic cells or directly damaged cells increased subsequent to exposure of the cells to capsaicin (P<0.001, Fig. 3).

An increase in cleaved caspase-3 expression was detected in the AGS cells following treatment with capsaicin (Fig. 4). Apoptosis is regulated by the balance between pro- and anti-apoptotic genes. As shown in Fig. 4, the protein levels of the anti-apoptotic gene Bcl-2 was reduced by treatment with capsaicin in AGS cells, whereas the protein levels of Bcl-xL were not altered in response to the treatment with capsaicin.

The effect of capsaicin on the MAPK signal pathway, which is essential for cell survival and growth during development and carcinogenesis, was examined. Capsaicin treatment decreased the expression of phosphorylated ERK 1/2 (Fig. 5). Since crosstalk between signal transduction pathways is common, we examined whether capsaicin prevented the phosphorylation of p38 or JNK in human gastric cancer cells. Capsaicin treatment slightly decreased the phosphorylation of p38 and JNK. The levels of total ERK 1/2, JNK and p38 MAPK were not altered after capsaicin treatment (Fig. 5).