Human GC cell line SGC-7901 was purchased from the Chinese Academy of Sciences, Shanghai Institute for Biological Science (Shanghai, China). SGC-7901 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum (both from GE Healthcare Life Sciences, HyClone Laboratories, Logan, UT, USA). The cultures were maintained at 37°C in 5% CO₂. Melatonin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was dissolved in ethanol prior to use. The final concentration of ethanol in the culture medium never exceeded 1%.

After exposure of GC cells to various concentrations of melatonin for various times, we assessed cell viability using the MTS assay (CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay; Promega, Madison, WI, USA), which is based on the mitochondrial conversion of MTS (a tetrazolium salt) into a water-soluble, colored, formazan precipitate that can be quantified by spectrophotometry. SGC-7901 cells were seeded at a density of 1×10⁴ cells/ml in 96-well plates. After 24 h of culture, the cells were treated with 0 (1% ethanol as control was added), 1, 2, 3, 4 or 5 mM melatonin for 24, 48 or 72 h. Absorbance of cells at 490 nm was measured using a microplate reader (Synergy HT; BioTek Instruments Inc., Winooski, VT, USA) when MTS was added 2 h later. Effects on SGC-7901 cell viability was measured by determining the percentage of viable cells relative to the control: % cell inhibition = [1 - (OD_mt - OD_blank)]/ (OD_c -OD_blank) × 100%, where OD_mt is the average OD value of the melatonin-treated samples, OD_c is the average OD value of the control samples, and OD_blank is the average OD value of the blank samples without cells.

SGC-7901 cells were seeded at a density of 2×10⁵ cells/ml in 6-well plates. After 24 h of culture, the cells were treated with 3 mM melatonin or 1% ethanol (control) for 24 h. Cell morphology was observed with an inverted microscope (Primo Vert; Carl Zeiss Microscopy GmbH, Jena, Germany) and an electron microscope (EM208; FEI, Hillsboro, OR, USA).

After treatment with melatonin, cells were collected and stained with propidium iodide (PI; BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA). Single-cell suspensions were sorted by flow cytometry, which revealed the distribution of cells in the three major phases of the cycle (G₀/G₁ vs. S vs. G₂/M).

We use the TUNEL assay (DeadEnd™ Fluorometric TUNEL System; Promega) to analyze cell apoptosis in situ. This assay measures nuclear DNA fragmentation of apoptotic cells tagged with fluorescein-12-dUTP, which can be visualized by fluorescence microscopy. SGC-7901 cells were cultured on coverslips and treated with either 3 mM melatonin or 1% ethanol for 24 h. After three washes with phosphate-buffered saline, the cells were fixed in 4% paraformaldehyde and incubated at 4°C for 25 min. Fixed cells were permeabilized with 0.2% Triton X-100 solution for 5 min. The cells were then incubated with a nucleotide mixture and rTdT buffer solution, and incubated at 37°C for 60 min to allow the tailing reaction to occur in the dark. After terminating the reaction in 2X SSC, the cells were counterstained with PI for 15 min at room temperature. Positive apoptotic cells were identified under 5 random fields of view by fluorescence microscopy (Axio Observer A1; Carl Zeiss Microscopy GmbH, Jena, Germany).

To analyze the rate of cellular apoptosis, we used the FITC Annexin V apoptosis detection kit I (BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA), following the manufacturer's instructions. Briefly, SGC-7901 cells were treated with 3 mM melatonin for 24 h and collected. In cells that have undergone apoptosis, phosphatidylserine (PS), which is usually located in the inner leaflet of the plasma membrane, is translocated to the outer leaflet of the plasma membrane. Once on the outer surface of the membrane, PS is bound by FITC-labeled Annexin V and detected by flow cytometry.

Melatonin-and vehicle-treated SGC-7901 cells were lysed in cell lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) supplemented with a protease inhibitor cocktail and phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentrations were measured using the enhanced BCA protein assay kit (Beyotime Institute of Biotechnology). Protein extracts (40 µg) were subjected to 12 or 15% polyacrylamide gel electrophoresis. The proteins in the gels were transferred to polyvinylidene difluoride membranes, which were then blocked in Tris-buffered saline containing 0.5% bovine serum albumin. Blocked membranes were incubated with primary antibodies: anti-MDM2 (cat. no. ab137413; dilution, 1:1,000), anti-phospho-MDM2 (at Ser166; cat. no. ab170880; dilution, 1:50,000), anti-CDC25A (cat. no. ab140247; dilution, 1:200), anti-phospho-CDC25A (at Ser75; cat. no. ab47279; dilution, 1:1,000), anti-p21 (cat. no. ab109199; dilution, 1:5,000), and anti-phospho-p21 (at Thr145; cat. no. ab47300; dilution, 1:1,000) were purchased from Abcam (Cambridge, UK); anti-AKT (cat. no. 4685S; dilution, 1:1,000), anti-phospho-AKT (at Thr308; cat. no. 4056S; dilution, 1:1,000), anti-Bcl-xL (cat. no. 2764S; dilution, 1:1,000), anti-Bax (cat. no. 5023P; dilution, 1:1,000), anti-caspase-9 (cat. no. 9508S; dilution, 1:1,000) and anti-GAPDH (cat. no. 2118S; dilution, 1:1,000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and anti-p53 was purchased from Medical and Biological Laboratories Co., Ltd., Nagoya, Japan (cat. no. K0181-3; dilution, 1:5,000). Proteins were detected by the addition of alkaline phosphatase-conjugated secondary antibody, goat anti-rabbit IgG (cat. no. ab98505; dilution, 1:5,000; Abcam) or goat anti-mouse IgG (cat. no. sc-2008; dilution, 1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Target proteins were visualized by the addition of CDP-Star reagents (Roche Diagnostics, Mannheim, Germany). The bands were detected using an ImageQuant LAS 4000 mini (GE Healthcare, Chicago, IL, USA). Band intensities were quantified using ImageJ2× software (National Institutes of Health, Bethesda, MD, USA) and the relative intensities to the internal GAPDH control were calculated.

SGC-7901 cells were seeded at a density of 1×10⁴ cells/ml in 96-well plates. After 24 h of culture, the cells were treated with 0 (1% ethanol as control) or 3 mM melatonin for 24 h. Melatonin- and vehicle-treated SGC-7901 cells were added together with 100 µl of Caspase-Glo^® 3/7 Reagent according to the caspase-Glo^® 3/7 assay kit (Promega Corp., Madison, WI, USA) manufacturer's instructions. This assay provides a luminogenic caspase-3/7 substrate that is released following caspase cleavage, and the subsequent production of light can be detected by a microplate luminometer (Orion microplate luminometer; Berthold Detection Systems GmbH, Pforzheim, Germany).

The data represent the means ± standard deviations (SD) from at least three independent experiments. One-way ANOVA and the Student's paired t-test were used to determine statistical significance. P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).

We assessed the cell viability using the MTS assay. Results demonstrated that cell growth was inhibited after melatonin exposure in a dose- and time-dependent manner (Fig. 1). Based on these results, we selected 3 mM melatonin and 24 h of exposure for the follow-up experiments; these parameters reflect a 50% inhibition of cell viability. Cells treated with higher concentrations of melatonin for longer times were not suitable for use in subsequent experiments. The morphology of treated SGC-7901 cells was assessed microscopically (Fig. 2). At the microstructural level, melatonin-treated cells appeared thinner and more elongated than control cells, with twisted cytoplasmic extensions and a greater number of floating dead cells. At the ultrastructural level, apoptotic cells were evident, surface microvilli were absent, plasmolysis and nuclear pyknosis were present, mitochondria exhibited vacuolation, and apoptotic bodies appeared in the cultures of melatonin-treated cells.

We assessed the distribution of SGC-7901 cells in various phases of the cell cycle by flow cytometry. The analysis showed that the proportion of cells in the G₀/G₁ phase increased from 59.357±1.518 to 68.583±0.649% (P<0.05) in the melatonin-treated SGC-7901 cells compared to the controls (Fig. 3). Consistent with this finding, the proportion of cells in the S and G₂/M phases decreased relative to the controls: As shown in Fig. 3, the proportion of S-phase cells decreased from 21.177±1.322 to 16.590±1.874% (P<0.05), and the proportion of G₂/M-phase cells decreased from 20.450±1.868 to 14.727±1.194% (P<0.01). All differences were statistically significant. These results indicate that melatonin effectively arrested SGC-7901 cells in the G₁/S phase of the cell cycle.

We used two methods to determine whether melatonin stimulates the apoptosis of SGC-7901 cells. First, we used the TUNEL assay to measure apoptotic SGC-7901 cells in situ. As shown in Fig. 4, apoptosis could be observed in the SGC-7901 cells treated with melatonin but not in the controls. We then used flow cytometry to determine the percentage of apoptotic cells using the FITC Annexin V apoptosis detection kit I. The percentages of melatonin-treated cells in the early and late stages of apoptosis were found to increase compared with the controls (Fig. 5). Early apoptotic cells increased from 0.97±0.31 to 7.25±3.00 (P<0.05), late apoptotic cells increased from 1.23±0.53 to 10.22±2.22 (P<0.05), and the total number of apoptotic cells increased from 2.20±0.81 to 17.48±4.98 (P<0.05). All differences were statistically significant.

CDC25A and p21 are known regulators of cell cycle progression that interact with cyclin/CDK complexes. Melatonin reduced the expression of CDC25A and the level of CDC25A phosphorylation at Ser75 in SGC-7901 cells compared to the controls (Fig. 6A). Moreover, we found that both the level of p21 and its phosphorylation at Thr145 were decreased (Fig. 6B). We examined changes in apoptosis-associated proteins Bcl-xL, Bax, caspase-9 and caspase-3. In SGC-7901 cells treated with melatonin, western blot analysis showed evidence of a reduced expression level of Bcl-xL and a significantly increased expression level of Bax compared with controls (Fig. 6C). Melatonin treatment also increased cleaved caspase-9 levels and caspase-3 activity (Fig. 6G and H).

MDM2, p53 and AKT are upstream regulators of the apoptosis- and cell cycle-related proteins mentioned above. To further understand the molecular mechanism underlying melatonin-induced cell apoptosis and inhibition of cell proliferation, we evaluated the expression levels of MDM2, p53 and AKT in SGC-7901 cells after melatonin exposure. Western blot analysis showed that the expression levels of MDM2 and phospho-MDM2 (at Ser166) were decreased compared with the controls (Fig. 6D), whereas the expression level of p53 was increased compared with the controls (Fig. 6F). Levels of both AKT and phospho-AKT (at Thr308) were decreased after melatonin treatment of SGC-7901 cells compared to the controls (Fig. 6E).