Figure 5.

Even though 5-FU induces phosphorylation of Akt and MAPKs in MDA-MB-231 cells and curcumin inhibits this upregulation, the synergism of 5-FU and curcumin is independent of both these survival signals. (a) Kinetics of 5-FU-induced activation of Akt in MDA-MB-231 cells after treating them with 5-FU for different time intervals (0–12 h). The whole cell lysate was immunoblotted using antibody against phospho-Akt (ser473) antibody and detected by enhanced chemiluminescent (ECL). β-Actin levels are shown as loading control. (b) Curcumin-mediated downregulation of 5-FU-induced activation of Akt. Western blot analyses were performed with anti-phospho-Akt (ser473) on whole cell lysates after 30 min of drug exposure. (c) Activation status of various MAPKs in MDA-MB-231 cells after exposing to 10 μM 5-FU for different time periods (0–12 h).The whole cell lysate was immunoblotted using phospho-specific antibodies against extracellular regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38. The expression level of β-actin is shown as loading control. (d) Downregulation of 5-FU-induced activation of various MAPKs in MDA-MB-231 cells by curcumin. Western blot analyses were performed using phospho-specific antibodies against the various MAPKs on cell lysates, after treating with indicated drugs for 30 min. (e) Dose-dependent activation of AP-1 by 5-FU in MDA-MB-231 cells. Nuclear extracts prepared from MDA-MB-231 cells after exposing them to different concentrations of 5-FU (0–25 μM) were assayed for AP-1 activation by electrophoretic mobility shift assay (EMSA). (f) Inhibition of 5-FU-induced activation of AP-1 by curcumin in MDA-MB-231 cells. Nuclear extracts prepared after exposing MDA-MB-231 cells to 5-FU and curcumin, either alone or in combination for a period of 1 h, were assayed for AP-1 activation by EMSA. (g) Supershift analysis, using anti-c-jun antibody to indicate band specificity, is carried out as described in Materials and Methods. (h) Effect of 5-FU and curcumin, alone or in combination, in MDA-MB-231 cells treated with Akt and MAPKs inhibitors. A total of 5000 cells in triplicates were pre-treated with curcumin, LY294002 (1 μM), U0126 (5 μM), SP600125 (5 μM) and SB203580 (1 μM), followed by 5-FU treatment for 48 h and subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data represent three independent sets of experiments and results are shown as the mean±S.D. ***, and ** represents P-values ⩽0.0001 and ⩽0.001 respectively. Inhibition status of Akt and various MAPKs were shown in inset