Figure 4

SXR activation increases expression of inducible nitric oxide synthase and raises NOS activity. A) MCF-7 and B) ZR-75-1 cells were grown in the presence of 10 μM concentration of SXR activators compounds rifampicin, anandamide or clotrimazole for 24, 48 and 72 hours and for 18 and 24 hours respectively. After the indicated time of treatment, total RNA was isolated, reverse transcribed and analyzed by QRT-PCR using primers for the human iNOS gene. Data are shown as average fold induction relative to solvent control in triplicates ± S.E.M. Results were replicated in at least three independent experiments.*represents P ≤ .05 and ** represents P ≤ .01 compared to control (by student's t test) C) SXR activation increase the levels of calmodulin protein in MCF-7 and ZR-75-1 cells. Equal amount of total cell lysates made from MCF-7 and ZR-75-1 cells treated with SXR activator compounds or solvent control for 24 and 18 hrs respectively were subjected to Western blot analysis using CaM antibody (C7055, sigma-aldrich) as described in materials and methods section. Equal loading was confirmed by probing with anti-GAPDH antibody. D) SXR activation increases iNOS activity. MCF-7 cells were activated by rifampicin or solvent control for 48 hours. Cytokine cocktail (IL1β (20 ng/ml), TNFα (15 ng/ml) and LPS(1 mg/ml) was used as a positive control. Nitric oxide synthase activity was determined intotal cells homogenates in the presence or absence of NOS inhibitors; L-NMMA and 1400 W (10 μM) as described in Materials and Methods. The data are depicted as counts per minute (CPM) per mg protein. The results have been replicated in independent runs. # represents P ≤ .001 in comparison to RIF (by student's t test) E) SXR activation leads to Nitric Oxide (NO) accumulation. MCF-7 cells were treated with SXR agonists (10 μM) or solvent control for 48 hours. The concentration of nitrate plus nitrite (stable metabolites of nitric oxide) in the culture supernatant of SXR agonists treated or control-treated MCF-7 cells was measured using the Griess method [36]. Data are depicted as average fold induction relative to solvent control in triplicates ± S.E.M. The results were replicated in at least three independent experiments. *represents P ≤ .05 compared to control (by one-way ANOVA analysis). F) L-NMMA and 1400 W, the nitric oxide synthase inhibitors block the increase in expression of p53 caused by rifampicin. MCF-7 cells were pre-treated with or without L-NMMA (500 μM) or 1400 W (10 μM) for 1 hr and then stimulated by rifampicin for 72 hours. Total RNA was isolated from the cells after completion of treatment and analyzed for p53 expression using QRT-PCR. The data are shown as average fold induction relative to solvent control in triplicates ± S.E.M. rifampicin, RIF; tamoxifen, TAM; anandamide, ANA; clotrimazole, CLO; camptothecin, CAMP. # represents P ≤ .001 and ** represents P ≤ .01 in comparison to RIF (by student's t test). rifampicin, RIF; anandamide, ANA; clotrimazole, CLO.