Figure 7

Effect of zoledronic acid (ZOL) on Cox-2 mRNA, protein levels and activity in MDA-MB-231 cells – Comparison with C3 exoenzyme. (A) Time course of ZOL on Cox-2 mRNA expression. MDA-MB-231 cells were treated for 3, 6 or 12 h with or without 1 μ M ZOL. RNA was extracted and analysed by RT–PCR. Sizes of PCR products for Cox-2 and GAPDH were, respectively, 300 and 222 bp. (B) Effect of ZOL on Cox-2 mRNA expression. MDA-MB-231 cells were incubated for 12 h with C3 exoenzyme (5 μg ml⁻¹) or with 1 μ M ZOL with or without geranylgeraniol (GGOH) (10 μ M) and farnesol (FOH) (10 μ M) and compared with untreated cells. RNA was extracted and analysed by RT–PCR. Sizes of PCR products for Cox-2 and GAPDH were, respectively, 300 and 222 bp. (C) Effect of ZOL on Cox-2 protein level. MDA-MB-231 cells were incubated with or without ZOL for 18 h and Cox-2 protein expression was examined by Western blot, using a monoclonal anti-Cox-2 antibody. Blots were developed with the enhanced chemoluminescence reagent (ECL). The membrane was also probed with β-actin to confirm equal loading. (D) Effect of ZOL on the secretion of PGE2 in the supernatant of MDA-MB-231 cells. The levels of PGE2 were measured by ELISA assay on supernatants of control and ZOL (1 μ M)-treated MDA-MB-231 cells in the presence or absence of GGOH and FOH. The effect of ZOL was compared with C3 exoenzyme. Results are mean±s.e.m. of three independent experiments and are expressed in picograms of PGE2 per microgram of protein (^**P<0.01).