Figure 3

Dickkopf-1 is regulated via inhibited geranylgeranylation. (A), (B) MDA-231 cells were treated with either zoledronic acid (ZOL; 100 μM) or atorvastatin (ATO; 10 μM) for 24 hours alone or concurrently with mevalonate substrates (geranyl-geranyl-pyrophosphate (GGPP), farnesyl pyrophosphate (FPP) or mevalonate (MEV)). Dickkopf-1 (DKK-1) was analyzed by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). (C) Specific inhibitors of geranylgeranylation GGTI-298 (5 μM) and farnesylation FTI-277 (100 nM) were used for further pathway clarification, and treatment was for 24 hours. Results of ELISA and PCR data are presented as the mean ± standard deviation of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Inhibition of geranylgeranylation by GGTI-298, atorvastatin and zoledronic acid, farnesylation by FTI-277, atorvastatin and zoledronic acid as well as their selective reversal by the substrates was confirmed by assessing unfarnesylated RAS and ungeranylated RAP1A in western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as loading control. Representative blots are shown. (D) Levels of phosphorylated β-catenin decrease following 24 hours of exposure to zoledronic acid and atorvastatin. Levels of total β-catenin remain unchanged. GAPDH is used as a loading control.