Apoptosis of Human Neuroblastoma Cells Induced by Liposome-Encapsulated Fenretinide

Abstract

Human neuroblastoma (NB) tumours represent a major therapeutic challenge due to the lack of drugs effective in controlling cell proliferation. We previously reported that the synthetic retinoid Fenretinide (HPR) inhibits NB cell growth through the induction of programmed cell death. More recently, various NB cell lines have been shown to be partially resistant, in vitro, to HPR used at in vivo achievable concentrations (1-3 μmol/L). To significantly increase the dose, half-life, and stability of this promising anticancer agent we studied a system of conventional or long-circulating liposomes.In this study, we showed that HPR can be efficiently and stable encapsulated in conventional (CL-HPR) and stabilized liposomes (SL-HPR). Since the leakage of the drug from the liposomes under the experimental conditions used is negligible, it seems that HPR is entering cells via uptake of intact liposomes. Liposome-entrapped HPR completely arrested the growth of NB cells. The effect was dose- and time-dependent. Indeed, SL-HPR at 30 (imol/ L induced, in the cell lines partially resistant to free HPR, a very rapid (24-48 h) fall in thymidine uptake (> 95 %), whereas at 3 μmol/L it exhibited cytostatic effects.Time lapse photomicroscopy showed that NB cells treated with SL-HPR underwent a death process highly reminiscent of apoptosis, with progressive condensation of the cytoplasm around the nucleus and intense cell shrinkage. The cells then rounded up and detached from the plate. Furthermore, propidium iodide staining of the DNA showed that a high proportion of cells treated with SL-HPR displayed a small and brightly staining nucleus; chromatin appeared aggregated into dense masses at the nuclear periphery, a typical feature of apoptotic cells. These findings were confirmed by electronic microscopy, DNA fragmentation assay, DNA content analysis and by a quantitative assay for evaluating programmed cell death based upon the labeling of DNA breaks with tritiated thymidine. HPLC analysis showed that HPR did not become metabolized after uptake into NB cells cultured in vitro, thus indicating that SL-HPR-induced apoptosis results from the action of HPR, itself, and not from its metabolite(s). In conclusion, our study demonstrates that Fenretinide entrapped in conventional or sterically stabilized liposomes dramatically suppresses NB cell growth by inducing programmed cell death.