HEC-1B and Ishikawa cell lines were obtained from the cell bank of the Chinese Academy of Science (Shanghai, China). Cells were all cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Darmstadt, Germany) or DMEM-F12 with 10% fetal bovine serum (FBS; Life Technologies, Darmstadt, Germany) and incubated in a humidified atmosphere of 5% CO₂ at 37°C.

Cell viability was determined by a colorimetric 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay in accordance with previously described protocols. Briefly, cells were plated in 96-well culture plates (2×10⁴ cells per well) then treated for 24, 48 and 72 h with various concentrations of curcumin. The cells were then washed twice with phosphate-buffered saline (PBS) and incubated with 5 mg/ml MTT (Sigma-Aldrich, St. Louis, MO, USA) for 4 h. Then, cells were washed with PBS and solubilized with dimethyl sulfoxide.

Cells were seeded into a six-well plate and cultured to 70% confluence in medium containing 10% FBS. Cell monolayers were scratched with a plastic tip (1 mm) and then incubated in serum-containing medium (1% serum) with 0, 10, 20 and 30 µM curcumin for 24 h. The migration distance of the cells was measured at three sites using Photoshop software (Adobe Systems, Inc., San Jose, CA, USA). The migration rate was expressed as a percentage of the control.

The invasion potential of the cancer cells was assessed in vitro using Transwell chambers (Corning Incorporated, Corning, New York, NY, USA). First, the upper chambers were coated with Matrigel (BD Biosciences, San Jose, CA, USA), then 1×10⁶ cells in serum-free medium were added to the upper chambers, and FBS (10%) was added to the bottom chambers. Cells on the bottom side of the filter were fixed, stained and counted. For transfection experiments, cells were seeded 24 h after transfection. The rate of invasive cells was expressed as a percentage of the control.

The expression plasmids were provided by Dr Chen and the transfection was performed as described previously (4). Briefly, the full-length pcDNA3.1 (Invitrogen Life Technologies, Carlsbad, CA, USA) MEK1 vector was prepared by cloning the full-length polymerase chain reaction product of MEK1 with KOD® DNA polymerase (Toyobo, Osaka, Japan). DNA sequencing was used to confirm the plasmid sequences. For transient transfection experiments, cells were plated 24 h prior to transfection in a six-well plate at a density of 2×10⁵ cells per well. Lipofectamine 2000 (Invitrogen) was used with 4.0 µg pcDNA3.1(+)-MEK1 vector or 4.0 µg pcDNA3.1(+) empty vector as a negative control in accordance with the manufacturer's instructions.

Following treatment with various concentrations of curcumin or U0126 (Sigma), 2×10⁶ cells were suspended in 200 µl lysis buffer (1 mmol/l EDTA, 40 mmol/l Tris-HCl, 150 mmol/l KCl, 1% Triton X-100, 100 mmol/l NaVO₃ and 1 mmol/l phenylmethylsulfonyl fluoride; pH 7.5). The proteins (80 µg) were separated by 10 or 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride membranes. After being blocked in defatted milk (5% in Tris-buffered saline with Tween-20 buffer) at 37°C, the membranes were incubated with various antibodies against MMP-2, MMP-9, extracellular signal-regulated kinase (ERK) 1/2, p-ERK1/2 or β-actin (all from Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. The membranes were then incubated with appropriate secondary antibodies for 1 h at room temperature. The bands were detected and expressed as arbitrary units. Densitometric analysis was performed using ImageQuant TL software (GE Healthcare, Chalfont, Bucks, UK).

The assay was performed as previously described (8). Following treatment with various concentrations of curcumin for 24 h, samples of conditioned cell media were collected and separated by 0.1% gelatin 8% SDS-PAGE electrophoresis. Then, the gels were washed twice in 2.5% Triton X-100 for 45 min at room temperature and then incubated in reaction buffer (40 mM Tris-HCl, 10 mM CaCl₂ and 0.01% NaN₃; pH 8.0) at 37°C for 14 h. The gels were stained with Coomassie brilliant blue R-250 gel stain. The intensities of bands on the gels were calculated using an image analysis system (Bio-Rad Laboratories, Richmond, CA, USA). The final volumes of the samples were adjusted by counting the viable cell number.

Experiments were repeated three times, and dates were analyzed using Student's t-test. All statistical tests and corresponding P-values were two-sided. P<0.05 was considered to indicate a statistically significant difference. Correlation analysis was performed using the Z-test.

The anti-proliferation effects of curcumin at various concentrations (0 to 100 µM) on HEC-1B cells are shown in Fig. 1. At 40 µM, curcumin significantly inhibited the proliferation of HEC-1B cells. At concentrations below 40 µM, the anti-proliferative effect was not evident. Thus, a concentration range of curcumin lower than this was selected for all subsequent experiments.

Since cell motility is a measure of the metastatic potential of cancer cells, the motility of HEC-1B cells was examined. As shown in Fig. 2A, a continuous rapid movement was observed in control cells. The movement of HEC-1B cells was significantly reduced following treatment with curcumin in a concentration-dependent manner; the inhibition rates were ~65.33, 69.23 and 90.07% at 24 h with 10, 20 and 30 µM curcumin, respectively (Fig. 2B). Fig. 2C reveals the effect of curcumin on the invasiveness of HEC-1B cells that were treated with 0, 10, 20 and 30 µM of curcumin for 24 h. Curcumin reduced the invasion of HEC-1B cells substantially in a concentration-dependent manner. A similar anti-metastatic effect of curcumin was observed in Ishikawa cells (results not shown). Quantification analysis indicated that the invasiveness of HEC-1B cells was reduced by 18.58, 51.89 and 73.58% when cells were treated with 10, 20 and 30 µM of curcumin (Fig. 2D), respectively. Curcumin also inhibits the invasion of Ishikawa cells (results not shown).

MMPs are crucial to cell invasion, so the expression of MMP-2/-9 in EC cells that were exposed to various concentrations of curcumin was measured. HEC-1B cells were treated with 0, 10, 20 and 30 µM curcumin for 24 h, and then subjected to western blotting. Fig. 3A and B reveal that curcumin significantly reduced the protein levels of MMP-2/-9 in a concentration-dependent manner compared with the control group in HEC-1B cells. Fig. 3C and D reveal similar results for the activity of MMP-2/-9 in HEC-1B cells.

To further investigate the mechanisms underlying the anti-metastatic effect of curcumin, western blotting was used to detect the expression of ERK1/2 and p-ERK1/2 in HEC-1B cells. Western blotting revealed that curcumin reduced the phosphorylation of ERK1/2 in a concentration-dependent manner (Fig. 4A and B).

To further analyze the exact effect of curcumin on the ERK pathway, we transfected HEC-1B cells with a plasmid (pcDNA3.1(+)-MEK1) expressing human MEK1 (Fig. 5D). Following transfection, activity of the ERK signaling pathway was increased (Fig. 5C). It was also observed that MEK1 reversed the inhibitory effect of curcumin on cell invasion (Fig. 5A and B). The reduction in the inhibition rate in the pcDNA3.1(+) group and the pcDNA3.1(+)-MEK1 group was ~78.4 and 64.92% following 24 h of treatment with 30 µM curcumin, respectively.

To further investigate whether the inhibitory effect of curcumin on cell invasion and MMP-2/-9 expression was correlated with inhibition of the ERK pathway, HEC-1B cells were pretreated with an ERK inhibitor (U0126; 10 µM) for 30 min and then incubated in the presence or absence of curcumin (10 µM) for 24 h. The cells with the indicated pretreatment were then subjected to in vitro invasion assay. The results reveal that treatment with U0126 and curcumin significantly reduced cell invasion (Fig. 4C and D) as well as MMP-2 and -9 protein expression (Fig. 4E and F).