Figure 3.

5-FU induces upregulation of TS, which is downregulated by curcumin, and inactivation of TS inhibits the synergism. (a) Kinetics of 5-FU-induced activation of TS in MDA-MB-231 cells at different time intervals (0–48 h). The whole cell lysate was immunoblotted against TS antibody and detected by enhanced chemiluminescent (ECL). β-Actin levels are shown as loading control. (b) Effect of curcumin on 5-FU-induced activation of TS. MDA-MB-231 cells were pre-treated with curcumin for 6 h and simultaneously exposed to 5-FU for 48 h and the whole cell lysate were immunoblotted against TS. (c) Small interfering RNA (siRNA)-mediated silencing of TS expression in MDA-MB-231 cells. Cells were transiently transfected with different concentrations of control and TS siRNA, and also checked for the expression of TS using western blotting. (d) Effect of 5-FU and curcumin, alone or in combination, on control and TS siRNA-transfected MDA-MB-231 cells. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described earlier in Figure 1a. Data represent three independent sets of experiments and results are shown as the mean±S.D. *** and ^Φ represents P-values ⩽0.0001 and >0.05 respectively. (e) Effect of curcumin and/or 5-FU on cleavage of poly (ADP-ribose) polymerase (PARP) in control and TS siRNA-transfected MDA-MB-231 cells. Western blotting was carried out using anti-PARP antibody. All the data are representative of three independent experiments