Cell culture and materials
Human pancreatic cancer cells (PANC-1, COLO-357, and ASPC-1) were obtained from Georgetown University’s tissue culture facility. All the cell lines were cultured as a monolayer in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 5% heat-inactivated fetal bovine serum and 25 μg/ml gentamicin (Invitrogen).
The following antibodies, reagents, and chemicals were obtained commercially: vitamin E succinate (VES) and monoclonal anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO); survivin antibody (Santa Cruz Biotechnology, Santa Cruz, CA); caspase-3, Cdc2, p53, p21, and cleaved PARP antibodies (Cell Signaling Technology, Danvers, MA); horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA); WST-1 reagent and protease inhibitor cocktail tablets (Roche Applied Science); ECL Plus Western blotting detection system (GE Life Sciences, Piscataway, NJ); and Coomassie protein assay reagent (Pierce Thermo Scientific, Rockford, IL).
The effects of VES on cell viability and the proliferation of pancreatic cancer cells were determined using a cell viability detection kit (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate, WST-1) according to the manufacturer’s instructions (Roche Applied Science, Indianapolis, IN). Briefly, pancreatic cancer cells (PANC-1, COLO-357, and ASPC-1) were seeded onto 96-well plates at a density of 3,000 cells per well in complete media and treated at various concentrations and times with a vehicle (ethanol) or VES. At the end of treatment, 10 μl of the WST-1 reagent was added to each well to measure cell proliferation using a plate reader (Bio-Rad Model 680) at A = 450/600.
Quantification of apoptosis by sub-G1 analysis
Apoptosis was determined by the flow cytometric analysis of cells treated with VES. The cells were treated with different concentrations of VES for the indicated time. The floating and adherent cells were pooled, washed, fixed in 70% ethanol and stained with propidium iodide for FACS analysis (Becton–Dickinson, Franklin Lakes, NJ). The percentage of cells in the sub-G1
phase was calculated using Reproman computer software and was used as an index of apoptosis as previously described (Kumar et al. 2004
Cell cycle analysis
To determine the effect of VES on the cell cycle, pancreatic cancer cells were plated in complete medium and allowed to grow to 70–80% confluence. The cells were synchronized by serum starvation for 18 h, treated with the indicated concentrations of VES for 24 h, washed, fixed with 70% ethanol, stained with propidium iodide as above and analyzed by FACS. Gating was set to exclude cell debris, doublets, and clumps.
DNA fragmentation assay
COLO-357 cells were treated with VES with indicated concentrations. DNA fragmentation was analyzed using the Apoptotic DNA Laddering Kit (Roche Applied Science). Apoptotic DNA fragments were isolated according to the manufacturer’s protocol and resolved on 2% agarose gels for visualizations of DNA ladders.
Transfection with siRNA
PANC-1 cells were plated in 6-well plates in complete medium and allowed to grow to 50% confluence. Approximately, 100 nM survivin or control siRNA (cell signaling) was transfected using the TransIT-siQUEST (Mirus, Madison, WI) transfection reagent according to the manufacturer’s protocol. At 24 h, indicated concentrations of VES or vehicle were added to the siRNA-transfected cells. Cells were lysed at 48-h post-transfection and subjected to Western blot analysis.
Immunoblotting was performed essentially as described previously (Kumar et al. 2004
). At indicated times after treatment with VES, adherent and floating cells were collected. Whole cell extracts (total cell homogenates) were prepared by lysing cells in radio immune precipitation assay buffer, and proteins were separated on a 4–12% gradient Tris–glycine SDS gel (Pierce). Following SDS–PAGE, proteins were transferred to polyvinylidene difluoride membranes, immunoblotted and detected using ECL reagent.
All the experiments were replicated at least 3 times. The data provided are a representative of the experiments performed. A Student’s t test was used to analyze treated versus untreated cells. Results are expressed as averages ±SD. P < 0.05 is considered significant.