Figure 2.

5-Fu-induced Src activation is mediated by reactive oxygen species (ROS). (a) ROS induce Src activation in colon cancer cells. After treatment with various doses of H₂O₂ for 30 min, cells were harvested. Phosphorylation of Src (Tyr416) and total Prx-SO₃ in whole-cell lysates were detected by western blot analysis. (b) Effect of 5-Fu on Src activation and ROS generation. HT29 and SW480 colon cancer cells were treated with 5-Fu (50 μM) for various times. Phosphorylated Src (Tyr416) and total Prx-SO₃ levels were determined by western blot analysis. Fold changes in Src activity after 5-Fu treatment are labeled below the western blot image and were calculated using the Image J (NIH) software program. (c) Generation of ROS with increasing concentrations of 5-Fu. HT29 and SW480 cells were treated for 48 h with 5-Fu at the dose indicated. Cells were then incubated for 30 min at 37 °C with 2.5 μM CellROX Green Reagent. FACS Calibur flow cytometry was used to detect ROS as described in Materials and Methods. (d) Effect of combining NAC or catalase and 5-Fu on Src activation. HT29 and SW480 colon cancer cells were pretreated with NAC (10 mM) or catalase (200 mU) for 2 h and then incubated for 24 or 48 h with 5-Fu (50 μM). Phosphorylated Src (Tyr416) and total Prx-SO₃ levels were determined by western blotting. β-Actin was detected in the same membrane and served as a loading control. Data shown are representative of results from triplicate independent experiments