FIGURE 4.

13 induces AR antagonism in PCa cells. (a) 13 inhibits DHT (1 nM)-stimulated AR transcriptional activity in MDA-kb2 cells. Cells were cultured in Leibovitz’s/L15 medium supplemented with charcoal-stripped FBS (10%) and were treated with drugs and DHT at indicated concentrations for 24 hr. Data reflect mean ± SD (n = 4). (b) C4–2 cells were cultured in RPMI-1640 medium supplemented with charcoal-stripped FBS (10%) and were treated with Enz and 13 at 2.5 and 5 μM in the presence of DHT (1 nM) for 6 days. Medium and drugs were refreshed every 2 days. Bars represent mean ± SD (n = 6–8). ***p < .001; ****p < .0001. (c) 13 downregulates FL AR and AR-V7. VCaP cells were treated with 13, 12b, SFN, Enz, and SAHA at indicated concentrations for 16 hr. Whole-cell lysates were subjected to Western blotting and probed with the indicated antibodies. (d) VCaP cells were treated with 13 (10 μM) for indicated time. AR, AR-V7, Hsp70, and HO-1 were analyzed using Western blotting and densitometry. (e) 13 reduces the stability of FL AR and AR-V7. VCaP cells were treated with cycloheximide (CHX, 25 μg/ml) in the absence or presence of 13 (10 μM). AR and AR-V7 proteins were analyzed using Western blotting and densitometry at indicated time points (2, 4, 8, 16 hr). (f) 13 downregulates transcription factors Sp1 and c-Myc. VCaP cells were treated with 13 at 5 or 10 μM for 24 hr. Sp1 and c-Myc proteins were analyzed using Western blotting. Representative images of Western blotting analysis are shown in c, d, and f. Relative expression levels normalized against GAPDH are indicated under blots. Veh, Vehicle; GAPDH, glyceraldehyde 3-phosphate dehydrogenase