Dickkopf-1 is regulated via Cdc42 and Rho in breast cancer. (A) MDA-231 cells were treated for 48 hours with Clostridium difficile toxin A (250 ng/ml) and assessed for Dickkopf-1 (DKK-1) expression. (B) MDA-231 cells were exposed to zoledronic acid (ZOL) or atorvastatin (ATO) alone or concurrently with a Rho/Rac/Cdc42 activator for 24 hours. Due to the relatively short half-life of the activator, cells were treated every 5 hours with 1 μg/ml of activator until cells were harvested after 15 hours. (C) MDA-231 cells were treated with a number of different GTPase inhibitors for 24 hours and assessed for DKK-1 expression. Concentrations used were 10 μM for Rho#1 and Rho#2, 100 μM for Rac#1, 10 μM for Rac#2 and 50 μM for the Cdc42 inhibitor. (D) MDA-231 cells were transfected with control single interfering RNA (siRNA) or two different siRNAs directed against Cdc42. Sufficient knockdown was verified by western blot. DKK-1 expression was analyzed 48 hours after transfection. (E) Regulation of DKK-1 by Cdc42 and Rho was verified at protein levels, by measuring DKK-1 levels in the supernatant of MDA-231 cells treated with the same inhibitors as in C. (F) The role of Rho signaling was further confirmed by measuring DKK-1 levels following 24 hours of Rho-associated protein kinase inhibition using Y-27632 and H-1152P. Results of enzyme-linked immunosorbent assay and polymerase chain reaction data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.