Figure 1.

The combination of sulforaphane and TRAIL is superior to single treatments in reducing self-renewal potential. (A) DU145 and PC3 cells were left untreated or were treated with 10 μM sulforaphane; after 24 h, TRAIL was added at a concentration of 5 ng/ml. After an additional 24 h, the nuclear proteins were harvested and DNA binding was analyzed by EMSA using a biotin-labeled oligonucleotide probe for the NF-κB promoter consensus sequence. The specific NF-κB shifts are marked. Competition with a 10-fold excess of unlabeled oligonucleotide (10 × C) served as a control for the binding specificity. (B) Twenty-four hours after TRAIL treatment, the cells were trypsinized and re-plated in a normal medium at a low density (500 cells/well) in 6-well plates. Ten days later, the cells were stained, and colonies containing >50 cells were counted under a dissecting Zeiss Stemi DV4 microscope. Images of the fixed and stained colonies are presented in the lower panel. The data are presented as the mean of three independent experiments, and SD are shown (^*p<0.05, ^**p<0.01). (C) Following in vitro treatment as described above, PC3 cells were transplanted onto the chorioallantoic membrane of fertilized chicken eggs at day 8 of embryonic development. Nine days later (day 17), the developed xenograft tumors were resected, and the tumor engraftment rates and tumor volumes were evaluated. The tumor volumes are presented as black dots and tumor engraftment is presented as the percentage of grown tumors relative to the number of each treatment group. Below the diagram, representative images show the transplantation of the tumor cells into a plastic ring on the CAM (left images), and a developed PC3 xenograft is marked with an arrow (right image).