Human GC cell lines, AGS and SNU638, were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and the Korean Cell Line Bank (Seoul, Korea), respectively. Cells were cultured in RPMI-1640 medium containing 25 mM HEPES and supplemented with 10% fetal bovine serum (FBS) (both from Hyclone, Logan, UT, USA), 50 U/ml penicillin, and 50 μg/ml streptomycin (Gibco, Grand Island, NY, USA). Cultures were incubated at 37°C in 5% CO₂ in a humidified environment. Cells were seeded on plates at a density such that they would be 40-50% confluent at the time of transfection. The Mcl-1-specific and control-scrambled siRNA duplexes were purchased from Bioneer (Daeheon, Korea) and Qiagen (Germantown, MD, USA), respectively. The siRNAs were transfected into cells using Lipofectamine^® RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Total RNA was isolated from the cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For each sample, 1 μg of total RNA was used to generate complementary DNA in a reaction containing 50 ng/μl oligo-dT (Promega, Madison, WI, USA) that was incubated at 72°C for 10 min. We then added MMLV transcription reagents (Promega) and RNAsin (Takara, Otsu, Shiga, Japan) to each reaction and incubated the samples at 42°C for 1 h and 72°C for 15 min. PCR amplification was performed using gene-specific primers and GoTaq^® DNA polymerase (Promega). The primers we used were specific for Mcl-1 (5′-TCC TCT TGC CAC TTG CTT TT-3′ and 5′-TGC TGG AGT AGG AGC TGG TT-3′); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 5′-ACC ACA GTC CAT GCC ATC AC-3′ and 5′-TCC ACC ACC CTG TTG CTG TA-3′). Amplicons were separated by electrophoresis on 1% (w/v) agarose gels containing ethidium bromide.

Proteins were extracted from the cells using RIPA buffer (1 M Tris-HCl, 150 mM NaCl, 1% Triton X-100 and 2 mM EDTA) supplemented with 1 mM PMSF, Halt™ Phosphatase Inhibitor Cocktail and Halt™ Protease Inhibitor Cocktail (both from Thermo, Rockford, IL, USA). Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin (BSA) buffer, PVDF membranes were probed with the appropriate primary antibody. We used antibodies against human Mcl-1, Snail, vimentin, E-cadherin, phosphorylated β-catenin, β-catenin, MEK1/2, phosphorylated ERK1/2, ERK1/2, p38 and phosphorylated p38 (all from Cell Signaling Technology, Danvers, MA, USA). Antibodies against human MMP-2, MMP-9, GAPDH and β-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against human CD44 and CD133 were purchased from R&D Systems (Minneapolis, MN, USA) and eBioscience (San Diego, CA, USA), respectively. Protein bands were detected using a chemiluminescent horseradish peroxidase substrate (Millipore) and an ImageQuant™ LAS 4000 Luminescence imager (Fujifilm, Tokyo, Japan). The density ratio (%) of protein bands was quantified using MultiGauge V3.2 image analyzer software (Fujifilm).

Cell adhesion assay was conducted by coating fibronectin (2 μg/ml; Calbiochem, La Jolla, CA, USA) and collagen type I and IV (40 μg/ml; Corning Inc., Corning, NY, USA), respectively. The coated wells were washed with PBS, and blocked with 0.2% BSA medium for 30 min. Then the transfected cell suspension was added into the coated wells and incubated for 1 h at 37°C. Non-adherent cells were removed by washing with PBS. The attached cells were reacted with WST-1 solution (Daeil Lab Inc., Seoul, Korea) medium at 37°C for 1 h. The optical density was measured at 450 nm. All experiments were carried out in triplicate.

We conducted cell invasion assays using Transwell chambers with 8-μm pores (Corning Inc.). Transwell chambers were coated with 1% gelatin in RPMI-1640 overnight and then allowed to dry at room temperature. Cells transfected with the Mcl-1 and scrambled siRNAs were resuspended in 120 μl of 0.2% (w/v) BSA solution and seeded into the upper chambers. The lower chambers contained 400 μl of 0.2% (w/v) BSA solution supplemented with 10 μg/ml human plasma fibronectin (Calbiochem) as a chemoattractant. After incubation for 24 h, the cells that had migrated to the bottom surface of the Transwell were fixed with 70% ethanol and stained with Diff-Quik solution (Sysmex, Kobe, Japan). The cells in the upper chambers were removed using a cotton tip. Stained cells in the lower chambers were counted using light microscopy, from five randomly selected fields of view (0.5×0.5 mm²).

Cell migration was measured using Ibidi Culture-Inserts (Ibidi, Regensburg, Germany). The cells transfected with the Mcl-1 and scrambled siRNAs were seeded on the culture-inserts and incubated at 37°C in a humidified environment. After 24 h, the culture-inserts were gently removed using sterile tweezers to create a cell-free gap. Cell migration into the cell-free gap was followed for 24 h, and photographed using an inverted microscope. The distance between gaps was normalized to 1 cm after images were captured at three random sites.

The associations between experimental groups were analyzed using a Student’s t-test. A value of P<0.05 was considered to indicate a statistically significant result.

To study the biological role of Mcl-1 in GC progression, we used siRNAs to knock down endogenous Mcl-1 expression in AGS and SNU638 cells. Expression levels of Mcl-1 mRNA and protein in all tested cells were reduced following transfection with the Mcl-1 siRNAs (Fig. 1). To investigate the relationship between Mcl-1 and EMT in the human GC cells, cell adhesion assays were performed. The cell adhesion ability was measured after transfection of the siRNAs using three cell adhesion substrates including fibronectin and collagen I and IV. The cell adhesion to fibronectin and collagen I was significantly increased in the Mcl-1 siRNA-transfected AGS (P=0.023 and 0.034, respectively) and SNU638 cells (P=0.045 and 0.025, respectively) compared to that of the scrambled siRNA-transfected cells (Fig. 2). To investigate phenotypic changes induced by EMT, the expression levels of EMT-associated genes (MMP-2, MMP-9, Snail, E-cadherin, and vimentin) were also assessed. We observed lower expression levels of vimentin, MMP-2, MMP-9 and Snail in the Mcl-1 siRNA-transfected AGS and SNU638 cells, compared to these levels in the scrambled siRNA-transfected cells. The E-cadherin expression level was increased in the Mcl-1 siRNA-transfected AGS cells, but this level was not significantly different in the SNU638 cells (Fig. 3). We investigated the possible effect of Mcl-1 on the expression of cancer stemness markers such as CD44 and CD133. CD44 and CD133 expression levels were unaltered by knockdown of Mcl-1 (Fig. 3). Our results indicate that Mcl-1 expression is associated with the induction of molecular and cellular alterations consistent with EMT.

For the cell migration assays, the artificial wound gap became significantly narrower for cells transfected with the control-scrambled siRNAs in comparison with that for the Mcl-1 siRNA-transfected cells at 12 and 24 h in the AGS cell line (P=0.001 and 0.019, respectively). Similar results were noted at 6 and 24 h for the SNU638 cell cultures (P=0.033 and 0.023, respectively, Fig. 4). For the cell invasion assays, 160.3±93.8 and 117.7±70.7 invading Mcl-1 siRNA-transfected AGS and SNU638 cells, respectively, were observed. In contrast, for cultures transfected with the scrambled siRNAs, 424.0±146.2 and 382.7±109.4 invading AGS and SNU638 cells, respectively were observed. These differences in invading cell numbers were significantly different (P=0.018 for AGS cells and P=0.009 for SNU638 cells, Fig. 5). Our findings indicate that Mcl-1 expression is required for GC cell migration and invasion, subsequently leading to tumor metastasis.

We assessed phos-phorylation levels of proteins in the β-catenin, MEK1/2 and MAPK signaling cascades using western blotting to determine their involvement in EMT regulation. The phosphorylation level of β-catenin was increased in the AGS and SNU638 cells when Mcl-1 was knocked down. Phosphorylation levels of MEK1/2 were decreased in the AGS and SNU638 cells when Mcl-1 expression was knocked down. Phosphorylation of ERK1/2 and p38 was decreased in the Mcl-1 siRNA-transfected SNU638 cells (Fig. 6).