Fig. 5

Sulindac induces ROS in colon cancer cells and reversal of sulindac sulfide-induced gene downregulation by thiol antioxidants. Sulindac sulfide increases cellular ROS level over time in SW480 and RKO cells. Sulindac sulfide decreases expression of Sp1, Sp3, Sp4, VEGF, bcl-2 and survivin and these effects are completely or partially reversed by antioxidants DTT, GSH and NAC cotreatment. Sulindac sulfide decreases expression of Bax in SW480 cells (reversible by antioxidants) but not in RKO cells. Sulindac sulfide also decreases expression of cyclin D1 and this is not reversible by antioxidants. a Cells were treated with 100 μM sulindac sulfide and cellular ROS level was measured by flow cytometry as described under Materials and Methods. Experiments were done in triplicate and results are expressed as fold of control (mean ± S.D.). Asterisk (*) indicates statistical difference between control (DMSO) and treatment (P < 0.05). b, c Cells were treated with DMSO, sulindac sulfide alone or in combination with antioxidants as indicated for 24 h. Antioxidants were pre-applied to cells for 45 min when used in combination with sulindac sulfide. Sp1, Sp3 and Sp4 cellular levels were determined by Western blot analysis. d, e VEGF, bcl-2, Bax, survivin and cyclin D1 were determined by Western blot analysis as described under Materials and Methods and β-actin was used as loading control