BxPC-3 and CFPAC-1 are adherent human pancreatic adenocarcinoma cell lines that were grown in RPMI 1640 and Iscove’s modified Dulbecco’s media, respectively. PANC-1 is a human epithelioid pancreas carcinoma cell line that was grown in Dulbecco’s modified Eagle’s medium (DMEM). All media were supplemented with 10% heat-inactivated fetal bovine serum, ref 1 2mM L-glutamine, 100��gml?1 streptomycin and 100Uml?1 penicillin. All PC cell lines were cultured at a constant temperature of 37��C in a humidified atmosphere of 5% carbon dioxide (CO2). The bisphosphonic acid monohydrate ZOL, 1-hydroxy-2-[(1H-imidazol-1-yl) ethylidene], a third-generation BP, was kindly provided as the hydrated disodium salt by Novartis Pharma AG (Basel, Switzerland).

The neutralised sodium salt of ZOL was dissolved in sterile ddH2O and used at a final concentration in a range of 1�C100��M. Stock solutions of ZOL were aliquoted and kept at ?20��C for long-term storage. Anti-caspase-9/-3 and anti-PARP MAbs were purchased from New England Biolabs (Beverly, CA, USA). Protein sepharose was purchased from Sigma (St Louis, MO, USA). Rabbit antisera raised against raf-1 C-12, ��actin, ERK-1 K-23 and ERK-2 MAb C-14 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-pan-ras MAb clone 10 was purchased from Calbiochem (San Diego, CA, USA). Anti-Akt MAbs, GSK3�� fusion protein and anti-pGSK3�� MAbs were all from Cell Signaling Tech. (Beverly, MA, USA). Cell proliferation assay Analysis of cell proliferation was performed on PC cells in the presence of increasing concentrations of ZOL by the MTT assay.

Briefly, PC cells (3 �� 104well?1) were seeded in 96-well plates in serum-containing media and allowed to attach for 24h. The medium was then removed and replaced with new medium containing ZOL at different concentrations. Cells were incubated under these conditions for a time course spanning 72h. Then cells were incubated with 10��lwell?1 of thiazolyl blue (MTT, 5mgml?1, Sigma) for 1h at 37��C. After incubation, 100��l of 0.04N HCl in isopropanol was added into each well and the absorbance was measured at a wavelength of 620nm in a microplate reader. A value of 100% was assigned to untreated control cultures, and the IC50 was defined as the concentration of drug that reduced the number of viable cells to 50% of the control cultures after 72h of exposure.

Flow cytometric analysis of apoptosis Brefeldin_A Apoptotic cell death was analysed by Annexin-V�CFITC staining and by propidium iodide (PI) detection systems. Annexin-V�CFITC binds to phosphatidylserine residues, which are translocated from the inner to the outer leaflet of the plasma membrane during the early stages of apoptosis. Labelling of apoptotic cells was performed using an Annexin-V kit (MedSystems Diagnostics, Vienna, Austria).