The new combination between the nucleoside analogue gemcitabine and the cholesterol-lowering drug fluvastatin was investigated in vitro and in vivo on the human pancreatic tumour cell line MIAPaCa-2. The present study demonstrates that fluvastatin inhibits proliferation, induces apoptosis in pancreatic cancer cells harbouring a p21ras mutation at codon 12 and synergistically potentiates the cytotoxic effect of gemcitabine. The pharmacologic activities of fluvastatin are prevented by administration of mevalonic acid, suggesting that the shown inhibition of geranyl-geranylation and farnesylation of cellular proteins, including p21rhoA and p21ras, plays a major role in its anticancer effect. Fluvastatin treatment also indirectly inhibits the phosphorylation of p42ERK2/mitogen-activated protein kinase, the cellular effector of ras and other signal transduction peptides. Moreover, fluvastatin administration significantly increases the expression of the deoxycytidine kinase, the enzyme required for the activation of gemcitabine, and simultaneously reduces the 5 0 -nucleotidase, responsible for deactivation of gemcitabine, suggesting a possible additional role of these enzymes in the enhanced cytotoxic activity of gemcitabine. Finally, a significant in vivo antitumour effect on MIAPaCa-2 xenografts was observed with the simultaneous combination of fluvastatin and gemcitabine, resulting in an almost complete suppression and a marked delay in relapse of tumour growth. In conclusion, the combination of fluvastatin and gemcitabine is an effective cytotoxic, proapoptotic treatment in vitro and in vivo against MIAPaCa-2 cells by a mechanism of action mediated, at least in part, by the inhibition of p21ras and rhoA prenylation. The obtained experimental findings might constitute the basis for a novel translational research in humans.

Background:The identification of molecular and genetic markers to predict or monitor the efficacy of bevacizumab (BV) represents a key issue in the treatment of metastatic colorectal cancer (mCRC).Methods:Plasma levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble VEGF receptor 2 (sVEGFR-2) and thrombospondin-1 (TSP-1) were assessed by ELISA assay at different time points in a cohort of 25 patients enroled in a phase II trial of GONO-FOLFOXIRI plus BV as first-line treatment of mCRC. VEGF: −2578A/C, −1498C/T, −1154A/G, −634C/G and 936C/T; and VEGFR-2: −604A/G, +1192C/T and +1719A/T, polymorphisms were assessed in a total of 54 patients.Results:Treatment with GONO-FOLFOXIRI plus BV determined a prolonged and significant reduction in plasma free, biologically active VEGF concentration. Interestingly, VEGF concentrations remained lower than at baseline also at the time of PD. Conversely, PlGF levels increased during the treatment if compared with baseline, suggesting a possible role in tumour resistance; moreover, sVEGFR-2 increased at the time of PD, as well as TSP-1. No association of assessed polymorphisms with outcome was found.Conclusion:Our study suggested the possible mechanisms of resistance to combined therapy in those patients with a progressive disease to be tested in ongoing phase III randomised studies.

Purpose: The aims of the present study were to evaluate the clinical activity and the pharmacodynamic profile of the novel schedule of a single i.v. standard dose of cyclophosphamide (CTX) immediately followed by an oral metronomic CTX regimen with celecoxib (CXB) and dexamethasone (DEX) in advanced hormone-refractory prostate cancer patients. Experimental Design: Twenty-eight patients (68% docetaxel-resistant) received 500 mg/m 2 CTX i.v. bolus on day 1 and, from day 2, 50 mg/day CTX p.o. plus 200 mg/twice a day CXB p.o. and 1 mg/day DEX p.o. until disease progression. Plasma vascular endothelial growth factor (VEGF) and thrombospondin-1 were detected by ELISA, and real-time reverse transcription-PCR of VEGF and thrombospondin-1 gene expression on peripheral blood mononuclear cell and of VE-cadherin (VE-C) in blood samples was done. Results: A confirmed prostate-specific antigen decrease of ≥50% from baseline was observed in 9 of 28 patients (32%). Median progression-free survival and overall survival were 3 months (95% confidence interval, 2.2-4.2 months) and 21 months (95% confidence interval, 12.4-29.4 months), respectively. Toxicity was mild and no grade 3 to 4 toxicities occurred. A significant relationship was found between plasma VEGF and prostate-specific antigen values (r = 0.4223; P < 0.001). VEGF levels significantly increased in nonresponders, whereas the responder patients maintained significantly lower levels of VE-C gene expression after the beginning of the treatment if compared with nonresponder ones. Conclusion: Metronomic CTX plus CXB and DEX showed favorable toxicity and activity profile in patients. VE-C gene expression and VEGF levels represent potentially useful pharmacodynamic markers for the clinical response.Metronomic oral cyclophosphamide (CTX) alone-the chronic administration of CTX at low doses with no prolonged drugfree breaks-and in association with antiangiogenic drugs is a promising clinical approach to metastatic breast and ovarian cancers (1, 2). The antitumor and antiangiogenic activity of metronomic CTX has been shown in various experimental models of human prostate cancer (3). Phase II clinical studies have obtained interesting results using metronomic oral CTX alone (50 mg/m 2 /day; ref. 4) or in combination with dexamethasone (DEX; 50 mg/day CTX plus 1 mg/day DEX; ref. 5) in hormone-refractory prostate cancer (HRPC) patients. Metronomic CTX was safe and well-tolerated, showing a prostatespecific antigen (PSA) of ≥50% decline in 69% of patients with a response duration of 8 months (5). The administration of metronomic CTX with drugs targeting angiogenesis has shown a high preclinical activity (6, 7). Celecoxib (CXB), a cyclooxygenase-2 inhibitor, has shown both antiangiogenic properties (8) and antitumor activity in a transgenic mouse model of prostate adenocarcinoma (9) with a marked decrease in vascular endothelial growth factor (VEGF) expression (10). Moreover, CXB

Metronomic chemotherapy refers to the administration of chemotherapy at low, nontoxic doses on a frequent schedule with no prolonged breaks. The aim of the study is to rationally develop a CPT-11 metronomic regimen in preclinical settings of colon cancer. In vitro cell proliferation, apoptosis and thrombospondin-1/vascular endothelial growth factor (TSP-1/VEGF) expression analyses were performed on endothelial (HUVEC, HMVEC-d) and colorectal cancer (HT-29, SW620) cells exposed for 144 h to metronomic concentrations of SN-38, the active metabolite of CPT-11. HT-29 human colorectal cancer xenograft model was used, and tumour growth, microvessel density and VEGF/TSP-1 quantification was performed in tumours. In vitro and in vivo combination studies with the tyrosine inhibitor semaxinib were also performed. SN-38 preferentially inhibited endothelial cell proliferation alone and interacted synergistically with semaxinib; it induced apoptosis and increased the expression and secretion of TSP-1. Metronomic CPT-11 alone and combined with semaxinib significantly inhibits tumour growth in the absence of toxicity, which was accompanied by decreases in microvessel density and increases in TSP-1 gene expression in tumour tissues. In vitro results show the antiangiogenic properties of low-concentration SN-38, suggesting a key role of TSP-1 in this effect. In vivo, the CPT-11 metronomic schedule is effective against tumour and microvessel growth without toxic effect on mice.

The antitumoral activity of two new pyrazolo[3,4-d]pyrimidine compounds (CLM3, CLM29) in vitro and CLM3 in vivo in DePTC has been shown, opening the way to a future clinical evaluation.