Diethylamine (DEA) and Nonidet P-40 inhibitor Sorafenib were obtained from ICN Biomedicals Inc. (Costa Mesa, CA, USA); mouse IgG1 anti-p21ras monoclonal antibody was purchased from Transduction Laboratories (Lexington, KY, USA), whereas anti-p21rhoA and anti-p42MAPK/ERK2 rabbit polyclonal antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated secondary antibodies and reagents for chemiluminescence detection of proteins in immunoblots were purchased from Amersham Life Science (ECL Western detection kit, Little Chalfont, UK). Universal Mount was from Research Genetics Inc. (Huntsville, AL, USA). Quantitative real-time polymerase chain reaction (PCR) reagents were purchased from Applied Biosystems (Foster City, CA, USA).

All other chemicals not listed in this section were obtained from Sigma Chemical Co. (St Louis, MO, USA). Fluvastatin (Novartis, Basel, Switzerland) and gemcitabine (Ely Lilly and Company, Indianapolis, IN, USA) were dissolved in sterile distilled water, then diluted in sterile culture medium immediately before their in vitro use, or in sterile saline solution for in vivo use. Plastic for cell culture was supplied by Costar (Cambridge, MA, USA). PD98059 was purchased from Calbiochem Biochemicals (Milano, Italy), dissolved in DMSO and diluted in culture medium. The CD nu/nu male mice, weighing 20�C25g, were supplied by Charles River (Milan, Italy) and were allowed unrestricted access to food and tap water.

Housing and all procedures involving animals were performed according to the protocol approved (approval number 11/04) by the ��Comitato di Ateneo per la sperimentazione animale’ (Academic Committee for the animal experimentation) of the University of Pisa, in accordance with the European Community Council Directive 86-609, recognised by the Italian government, on animal welfare and the guidelines of the UK Co-ordination Committee on Cancer Research (UKCCCR). Each experiment employed the minimum number of mice needed to obtain statistically meaningful results. Cell culture conditions The human pancreatic cancer cell line MIAPaCa-2 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MIAPaCa-2 cells were maintained in DMEM medium, supplemented with 10% FBS, 2.5% HS, penicillin (50IUml?1), streptomycin (50��gml?1) and L-glutamine (2mM).

The human colon cancer cell line COLO320-DM, a cell line with wild-type K-ras (Di Paolo et al, 2000), was from ATCC and maintained in RPMI 1640 medium supplemented with 10% FBS, penicillin and L-glutamine. Cells were routinely grown in 75cm2 tissue culture flasks and kept in a humidified atmosphere of 5% CO2 at 37��C. Cells Cilengitide were harvested with a solution of 0.25% trypsin�C0.03% EDTA when they were in log phase of growth, and maintained at the above-described culture conditions for all experiments.