BMC Cancer 2004, 4:63 http://www.biomedcentral.com/1471-2407/4/63 gSnpFcriiuaghomdeucimersenausttl2piwechliirtachetietipnersdeloispbleuoyntsitohamnteioecnso-boeyfntmchaepasgnueslmaotcifoitnahbeoinfpeare2esn5ec0nacmpesMuolfaatamiopmnHo- Schematic representation of the gemcitabine encapsulation process within liposomes by means of the presence of a pH gradient elicited by the co-encapsulation of a 250 mM ammo- nium sulphate solution. the extravasation of liposomes, with a consequent accumulation of anticancer drugs in the site of action [22]. Furthermore, it should be considered that liposomes are non-toxic being mostly constituted by naturally occurring lipids. In the last years, gemcitabine has emerged as a very potent anti-tumour drug and it is currently used, alone or in com- bination, in the treatment of patients with different malig- nancies, including ovarian, pancreatic, non-small cell lung and other cancers [3-6]. Despite its promising effec- tiveness derived from a preclinical study [8], no apprecia- ble response was observed in a clinical study including four patients with aggressive thyroid anaplastic carcinoma treated with combination of gemcitabine and vinorelbine [9]. Although low, compared to other anti-neoplastic drugs, hematological and other toxicities appeared when effective anti-tumour doses of gemcitabine were tested. In this study, we prepared a liposome formulation with a high entrapment efficiency of gemcitabine and tested its efficacy against a well established human poorly differen- tiated thyroid carcinoma cell line. Our data demonstrate that, in ARO cells, incorporation of gemcitabine in lipo- somes enhances the cytotoxic effect of the drug as com- pared to free gemcitabine, suggesting a more effective uptake of the drug inside the cells. This activity was dem- onstrated at concentrations even lower than serum levels obtained in clinical trials. In our knowledge, no data are available in the literature concerning the relationship between gemcitabine uptake and its cytotoxicity, since FCiygtuortoex3ic effect of gemcitabine-loaded liposomes Cytotoxic effect of gemcitabine-loaded liposomes. ARO cells were grown to 50% confluence and treated with increasing concentrations of either free gemcitabine or gemcitabine- loaded liposomes for 24 h. Cell mortality was evaluated by trypan bleu dye exclusion assay (see Methods). The values, expressed as the ratio over control, represent the mean ± SEM of at least three different experiments in triplicates. neither liposomes or other carrier system have been tested against tumour cells. Our preliminary experiments (unpublished observations) show that a similar effect (improved uptake and cytotoxicity at lower doses) may be obtained also in colon cancer cells. It is note worthing that the various component used for the preparation of the liposomal delivery device were already approved by regu- latory offices to be used in the preparation of liposome- based formulations, i.e. Doxil®. Conclusion The clinical predictive value of the in vitro cell line pre- clinical cancer model is well established [23] and the ARO cells are one of the cell model of anaplastic thyroid cancer more exploited both for investigating the mechanism of tumour progression and for testing new molecules with antiproliferative effect. However, these promising obser- vations need to be confirmed in studies using in vivo xenograft cancer models, prior to propose the use of such Page 4 of 5 (page number not for citation purposes)