Figure 2

SXR activation reduces proliferation of breast cancer cells. A) MCF-7 and ZR-75-1 cells were grown in the presence of 1, 10 or 100 μM concentrations of SXR activators. Rutin, an isoflavone that does not activate SXR, and DMSO solvent were used as negative controls. Cell proliferation was measured after seven days using a fluorescence assay wherein emission at 535 nm is proportional to cell number. The values represent the average emission for triplicates ± S.E.M. These results were replicated in independent experiments. ** represents P ≤ .01 and # represents P ≤ .001 in comparison to solvent control (by one way ANOVA analysis) B) SXR activators cause G0/G1 arrest as compared to DMSO solvent controls. MCF-7 cells treated with 10 μM SXR activators for 24 hours were fixed, stained with propidium iodide and data acquired on FACSCalibur (BD Biosciences). Data were analyzed by CellQuest (BD Biosciences) and FlowJo (Tree Star, San Carlos, CA) software. Modfit (Verity Software, Topsham, ME) software was used to quantitate cell cycle using the fluorescence values of the FL2-area channel. The experiment was repeated twice in duplicates and the numbers represent the average of four values. Note: to measure the cell cycle distribution the population was live gated. C) SXR activators cause apoptosis in MCF-7 cells starting at 48 hours post-treatment. MCF-7 cells were grown in the presence of 10 μM SXR activators rifampicin, anandamide, or clotrimazole (RIF, ANA or CLO). Apoptosis was measured at 24, 48 and 72 h in the cytoplasmic fraction using the Cell Death Detection ELISA (Roche Applied Science, Germany). DMSO treatment was used as a negative control and 24 hour camptothecin (CAMP) treatment was used as a positive control for apoptosis. The values represent average of triplicates ± S.E.M and the results were replicated in two independent experiments. ** represents P ≤ .01 and # represents P ≤ .001 (by one way ANOVA analysis).