Bisphosphonates (BPs) are an emerging class of drugs mostly used in the palliative care of cancer patients. We investigated the in vitro activity of the most potent antiresorptive BP, zoledronic acid (ZOL), on the growth and survival of three human pancreatic cancer (PC) cell lines (BxPC-3, CFPAC-1 and PANC-1). Pancreatic cancer frequently has a dysregulated p21 ras pathway and therefore appears to be a suitable target for BPs that interfere with the prenylation of small GTP-binding proteins such as p21 ras . We found that ZOL induces growth inhibition (IC 50 :10 -50 mM) and apoptotic death of PC cells. The proapoptotic effect was correlated to cleavage/ activation of caspase-9 and poly(ADP)-ribose polymerase, but not of caspase-3. Moreover, we studied the p21 ras signalling in cells exposed to ZOL and detected a reduction of p21 ras and Raf-1 content and functional downregulation of the terminal enzyme ERK/ MAPkinase and of the pKB/Akt survival pathway. Finally, we observed that ZOL induces significant cytoskeletal rearrangements. In conclusion, we demonstrated that ZOL induces growth inhibition and apoptosis on PC cells and interferes with growth and survival pathways downstream to p21 ras . These findings might be relevant for expanding application of BPs in cancer treatment.

Pamidronate (PAM) and zoledronic acid (ZOL) are aminobisphosphonates (BPs) able to affect the isoprenylation of intracellular small G proteins. We have investigated the antitumor activity of BPs and R115777 farnesyl transferase inhibitor (FTI) against epidermoid cancer cells. In human epidermoid head and neck KB and lung H1355 cancer cells, 48 h exposure to PAM and ZOL induced growth inhibition (IC 50 25 and 10 lM, respectively) and apoptosis and abolished the proliferative and antiapoptotic stimuli induced by epidermal growth factor (EGF). In these experimental conditions, ZOL induced apoptosis through the activation of caspase 3 and a clear fragmentation of PARP was also demonstrated. A strong decrease of basal ras activity and an antagonism on its stimulation by EGF was recorded in the tumor cells exposed to BPs. These effects were paralleled by impaired activation of the survival enzymes extracellular signal regulated kinase 1 and 2 (Erk-1/2) and Akt that were not restored by EGF. Conversely, farnesol induced a recovery of ras activity and antagonized the proapoptotic effects induced by BPs. The combined treatment with BPs and R115777 resulted in a strong synergism both in growth inhibition and apoptosis in KB and H1355 cells. The synergistic activity between the drugs allowed BPs to produce tumor cell growth inhibition and apoptosis at in vivo achievable concentrations (0.1 lmolar for both drugs). Moreover, the combination was highly effective in the inhibition of ras, Erk and Akt activity, while farnesol again antagonized these effects. In conclusion, the combination of BPs and FTI leads to enhanced antitumor activity at clinically achievable drug concentrations that resides in the inhibition of farnesylationdependent survival pathways and warrants further studies for clinical translation.

Interferon alpha (IFN-alpha) has been widely used in the treatment of human solid and haematologic malignancies. Although the antitumour activity of IFN-alpha is well recognised at present, no major advances have been achieved in the last few years. Recent findings have provided new information on the molecular mechanisms of the antitumour activity of the cytokine. In fact, IFN-alpha appears to block cell proliferation, at least in part, through the induction of apoptotic effects. This cytokine can also regulate the progression of tumour cells through the different phases of the cell cycle inducing an increase of the expression of the cyclin-dependent kinase inhibitors p21 and p27. However, it must be considered that IFN-alpha is a physiologic molecule with ubiquitously expressed receptors that is likely to activate survival mechanisms in the cell. We have recently identified an epidermal growth factor (EGF) Ras-dependent protective response to the apoptosis induced by IFN-alpha in epidermoid cancer cells. The identification of tissue- and/or tumour-specific survival pathways and their selective targeting might provide a new approach to improve the efficacy of IFN-alpha-based treatment of human cancer. Moreover, new pegylated species of IFN-alpha are now available with a more favourable pharmacokinetic profile. We will review these achievements, and we will specifically address the topic of IFN-alpha-based molecularly targeted combinatory antitumour approaches.

Summary Valproic acid (VPA) is a well‐tolerated anticonvulsant that exerts anti‐tumour activity as a histone deacetylase inhibitor. This study investigated the in vitro and in vivo activity of VPA against multiple myeloma (MM) cells. In vitro exposure of interleukin‐6‐dependent or ‐independent MM cells to VPA inhibited cell proliferation in a time‐ and dose‐dependent manner and induced apoptosis. In a cohort of severe combined immunodeficiency mice bearing human MM xenografts, VPA induced tumour growth inhibition and survival advantage in treated animals versus controls. Flow cytometric analysis performed on MM cells from excised tumours showed increase of G0–G1 and a decreased G2/M‐ and S‐phase following VPA treatment, indicating in vivo effects of VPA on cell cycle regulation. Gene expression profiling of MM cells exposed to VPA showed downregulation of genes involved in cell cycle progression, DNA replication and transcription, as well as upregulation of genes implicated in apoptosis and chemokine pathways. Pathfinder analysis of gene array data identified cell growth, cell cycle, cell death, as well as DNA replication and repair as the most important signalling networks modulated by VPA. Taken together, our data provide the preclinical rationale for VPA clinical evaluation as a single agent or in combination, to improve patient outcome in MM.

Interferon a (IFNa) induces both apoptosis and a counteracting epidermal growth factor Erk-dependent survival response in cancer cells. In this report, IFNa increased eukaryotic elongation factor 1A (eEF-1A) protein expression by inhibition of eEF-1A degradation via a proteasome-dependent pathway. The reduction of the expression level of eEF-1A by RNA interference enhanced the apoptosis induced by IFNa on the same cells. Moreover, IFNa induced the phosphorylation of both serine and threonine in eEF-1A. These effects were paralleled by an increased co-immunoprecipitation and colocalization of eEF-1A with C-Raf. The suppression of C-Raf kinase activity with the inhibitor BAY 43-9006 completely antagonized the increase of both eEF-1A phosphorylation and expression and of C-Raf/eEF-1A colocalization induced by IFNa and enhanced apoptosis and eEF-1A ubiquitination. Cell transfection with the mutated K48R ubiquitin increased EF-1A expression and desensitized tumor cells to the modulating effects of IFNa. The dynamic simulation of 3Dstructure of eEF-1A identified putative serine and threonine phosphorylation sites. In conclusion, the interaction between eEF-1A and C-Raf increases eEF-1A stability and induces a survival activity.