It has similar efficacy and more info tolerability profiles than other HMG-CoA reductase inhibitors, but, unlike them, it has not been associated with rhabdomyolysis and myopathy (Scripture and Pieper, 2001). Few reports have been focused on fluvastatin inhibition of cancer cell proliferation (Seeger et al, 2003) and its possible mechanism of action (Kusama et al, 2001, 2002). To our knowledge, no combination studies have been performed with currently available chemotherapeutic drugs in order to test a hypothetic synergism with their anticancer activity and the molecular basis of the eventual positive interaction. The cytosine arabinoside analogue 2��,2��-difluorodeoxycytidine (gemcitabine) has been proven to be active in the treatment of pancreatic cancer (Abbruzzese, 2002a) with significant clinical benefit, but still with marginal survival advantage (El Rayes et al, 2003).

Gemcitabine inhibits cell growth by interfering with the synthesis of DNA (Barton-Burke, 1999), and efforts are currently being made to increase the therapeutic efficacy of the drug in clinical settings on various types of cancer by combination with other agents, including cisplatin, oxaliplatin, irinotecan, docetaxel, 5-fluorouracil, capecitabine or pemetrexed (Heinemann, 2002; Jacobs, 2002). Moreover, numerous preclinical experimental studies have been made to enhance the antitumour effects of gemcitabine using novel therapeutic approaches such as the proteasome inhibitor bortezomib (Kamat et al, 2004) and the antiangiogenic drug SU5416 (Bocci et al, 2004).

Gemcitabine and fluvastatin do not have overlapping toxicities; therefore, the association of these compounds might be an attractive clinical alternative for the treatment of advanced pancreatic tumours. The purposes of this study are to determine: (1) the antiproliferative, proapoptotic effects of fluvastatin on Brefeldin_A k-ras-mutated MIAPaCa-2 human pancreatic cancer cells and its probable mechanism of action; (2) the synergistic enhancement of cytotoxicity by the combination with gemcitabine; (3) the possible underlying molecular basis of the synergism with pharmacological tools such as PD98059, a MEK1/2 inhibitor that can block activation of downstream ERK-1/2, and the expression of genes such as the deoxycytidine kinase (dCK), a rate-limiting enzyme required for the activation of gemcitabine, and the 5��-nucleotidase (5��-NT), responsible for deactivation of gemcitabine; (4) the in vivo effects of the fluvastatin/gemcitabine combination on MIAPaCa-2 xenografts in nude mice. MATERIALS AND METHODS Materials and animals Antipain, leupeptin, aprotinin, sodium dodecyl sulphate (SDS) and proteinase K were obtained from Roche Molecular Biochemicals (Mannheim, Germany).