Alcohol stimulates activation of Snail, epidermal growth factor receptor signaling, and biomarkers of epithelial-mesenchymal transition in colon and breast cancer cells.

Alcohol stimulates upregulation of the signature EMT mesenchymal marker protein vimentin in breast and colon cancer cells and in normal intestinal epithelial cellsThe human cell lines Caco-2 (colon cancer)(Figure 1A) and MDA-MB-231(Figure 1B), and MCF-7 (Figure 1C)(both breast cancer) as well as the rat normal intestinal epithelial cell line IEC-6 (Figure 1D) were grown on glass coverslips in 6-well plates in complete medium. Treated cells were stimulated with alcohol (0.2%, 43mM) for 4 days and then Control (A, no alcohol) and alcohol-treated (B) cells were fixed and stained with Ab to vimentin and Alexa fluor 488 (green) fluorescent secondary Ab. Images (40x oil immersion) are representative from duplicate wells in three experiments. Arrows note increased cytoplasmic vimentin staining. Scale bar is 20 μm.

Alcohol stimulates production of MMP-2, MMP-7, and MMP-9 characteristic for EMT2A–2-C: The cell lines Caco-2, MDA-MB-231, and IEC-6 (all 1 × 106 cells/well) were cultured in 6-well plates and made serum free for 24h before stimulation overnight (24h) with alcohol (0.2%, 43 mM) in 1 ml of serum free media. Media was removed and 50μl/lane from control (no alcohol) and alcohol-treated cells was loaded onto SDS PAGE gels and MMP proteins were determined by western blot with Abs to MMP-2, MMP-9, and MMP-7 (lower). 2D: Caco-2 cells were treated as above for 24h with differing doses of alcohol (0.1% 22 mM-2% (434 mM)) and MMP-7 measured by western blot as above. Far right “+C” lane is positive control for MMP-7. (densitometry histogram: ProMMP-7=black, activated MMP-7=grey). All blots are representative of three experiments. * p ≤ .05 for all figures versus control.

Alcohol stimulates phosphorylation of the EMT transcription factor Snail at Ser246 and its localization to the nucleus3A–3D: Equal cell numbers of Caco-2, MDA-MB-231, MCF-7, and IEC-6 cells were stimulated with alcohol (0.2% (43mM)) and nuclear extracts prepared after 30m, 1h, 2h, 4h, and 6h. Aliquots equalized for protein were analyzed by western blot with Ab to Snail p-Ser246 and Histone H3 to measure loading. Blots are representative from 4 experiments while the densitometry data shown represents the means of all 4 experiments for each cell type so some bars are slightly different from the blots shown.

Figure 4A. Alcohol stimulates increased mRNA expression from a Snail reporter plasmid and repressed mRNA expression from an E-cadherin reporter. Caco-2 cells in complete medium in 96-well plates were transfected with 100ng/well of the Snail-PGL3 reporter plasmid containing the human Snail promoter (−869/+59) or the CDH1::LUC E-cadherin reporter (−308/+21) as well as a control renilla luciferase plasmid (1ng/well). Cells made serum free for 24h were stimulated with alcohol (0.2% (43 mM)) for 8h for the Snail reporter data or for 24h and 48h for the E-cadherin data. Firefly luciferase values were determined and normalized to renilla luciferase values to control for transfection efficiency. Data (% Control) are means of duplicate wells from 3 experiments (N=6).Figure 4B–4C. Alcohol stimulates mRNA expression of Snail and Snail protein expression in colon and breast cancer cells. Caco-2 colon cancer and MDA-MB-231 breast cancer cells grown in 6-well plates in complete medium were made serum free for 24h and then stimulated for 24h with alcohol (0.1% (22 mM), 0.2% (43 mM), or 1% (217 mM)). RNA was prepared and Snail mRNA expression normalized to β-actin by qRT-PCR analysis. Data are means from a representative of three experiments (upper sections). In addition cell lysates from matched wells were analyzed by western blot for total Snail protein with a representative blot shown from three experiments (lower sections).Figure 4D. Snail mRNA expression is significantly elevated in the colonic mucosa of heavy drinkers. Pinch sigmoid mucosal biopsies were obtained at 20 to 25 cm from the anus during sigmoidoscopy procedure (N=4/group). Controls were healthy subjects who had never been a daily or heavy drinker. Alcoholic subjects were selected based on NIAAA and DSM-IV criteria. RNA was prepared and Snail mRNA expression determined by qRT-PCR and data are expressed relative to β-actin.

SiRNA knockdown of Snail prevents alcohol induced vimentin expressionCaco-2 cells were treated with either media alone (lane 1), EtOH alone (lane 2), EtOH with control (scrambled) siRNA (lane 3), or EtOH with siRNA specific for Snail (lane 4). After 48h cells were then treated with alcohol for 4 days as in Figure 1 and then vimentin and total Snail proteins were determined by western blot. Blot is a representative from three experiments, histogram shows summarized data from all 3 experiments.

Figure 6A–6B. Alcohol stimulates transactivation of the EGFR and ERK1/2 signaling. Caco-2 and MDA-MB-231 cells made serum free for 24h were stimulated with alcohol (0.2%, 43mM) for 10 min for p-EGFR and 30 min for the p-ERK1/2 analysis. Some cells were preincubated with an inhibitor of the EGFR (AG1478, 500μM) or TACE (TAPI-2, 20μM). Western blots of cell lysates were developed with antibody to phospho-EGFRTyr1068 or phospho-ERK1/2 for Caco-2 cells (Figure 6A) and for MDA-MB-231 cells (Figure 6B). Blots were stripped and reprobed with Ab to total EGFR or ERK1/2 to show equal loading. Blots are representative of 3 experiments.Figure 6C. Alcohol stimulates translocation of TACE to the cell surface in colon and breast cancer cells. Caco-2 and MDA-MB-231 cells grown on glass coverslips were stimulated with alcohol (0.2%, 43 mM) for 30 min. Cells were then fixed and permeabilized and stained with Ab to TACE or ZO-1 and then fluorescent secondary Abs to TACE (red) or ZO-1 (green). 20x 1μm z-stack images were taken (40x oil immersion) and deconvoluted with Zeiss Axiovision software to yield the 3D images shown. Images are representative from three experiments. Bars =20μm.Figure 6D. Alcohol stimulates EGFR-dependent migration of breast cancer cells. MDA-MB-231 breast cancer cells in serum free medium (5 × 105 cells/well) were added to the upper well of 12-well/ 8 μm format PET transwells. Then 100ng/ml TNF-α (T) was placed in the bottom well and migration measured after 24h. For some wells ethanol (E, 0.2%, 43 mM) was added before adding cells ± specific inhibitors of the EGFR (AG1478, 500μM); TACE (TAPI-2, 20μM); or ERK1/2 (MEK)(PD98059, 30μM). Data are means of cells per hpf (40x) from duplicate wells of a representative of three experiments. [*=p ≤.05 for lane 2 vs. lane1, lane 3 vs. lane 2, and lanes 4,5,6 vs. lane 3].