Figure 3

Muscadine grape extract (MSKE) and SOD abrogates Snail-mediated superoxide species in LNCaP and ARCaP cells and reduces cell migration. (A) LNCaP-Snail high, ARCaP-Snail med and ARCaP-Snail high cells were plated overnight, serum starved for 3 h followed by treatments with either 5 μg/ml MSKE, 20 μg/ml MSKE, or 500 u/ml SOD for 3 days. Ethanol (EtOH) treated cells Neo- or Snail-transfected cells were utilized as controls. To assay for superoxide levels, whole cell lysates were prepared and 100 μl incubated with 25 μM Hydro-Cy3 for 15 min. OD was measured at 530/590 nm and normalized to protein concentrations. (B) LNCaP-Snail high or (C) ARCaP-Snail med was treated with 5 μg/ml MSKE, 20 μg/ml MSKE or 500 U/ml SOD for 3 days. Cells were then trypsinized and 50,000 cells plated on collagen-coated boyden chambers overnight. For LNCaP cells, since their cell migratory potential is low, a serum gradient was applied with 0.1% serum in the top chamber and 10% serum in the bottom chamber. For ARCaP cells, the top and bottom chamber both contained 0.1% serum. Cells that had migrated to the underside of the cell insert were solubilized with Sorenson solution and OD assayed at 590 nm to obtain relative cell migration. Results are representative of triplicate experiments performed independently, with data reported as mean ± SD (* p < 0.05; **p < 0.005, compared to EtOH control (-) Snail-transfected cells).