c-Met antisense oligodeoxynucleotides as a novel therapeutic agent for glioma:: In Vitro and In Vivo studies of uptake, effects, and toxicity

Background. c-Met, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor, are critical in cellular proliferation, motility, and invasion and are known to be overexpressed in gliomas. The aim of our study was to investigate the uptake and effects of c-Met antisense oligodeoxynucleotides (ASODNs) on rat and human glioma cells in vitro and the uptake and toxicity of these nucleotides in rat carcinomatosis and brain tumor models. Materials and methods. The three human cell lines (U87, BT325, SHG44) and the C6 rat glioma cell line were cultured. To study the uptake of oligodeoxymicleotides (ODNs) by glioma cells in vitro, cultured glioma cells readily incorporated caroboxyfluoreseein-5-succimidyl ester (FAM) labeled phosphorothioate oligodeoxynucleotides, as demonstrated by immunofluorescence microscopy and flow cytometry. To study the effect of ASODNs treatment on c-Met expression in vitro, Expression of c-Met was assessed by immunofluorescence microscopy and reverse transcriptase polymerase chain reaction (RT-PCR) analysis. For animal studies of ODNs toxicity and uptake, eight rats underwent placement of cisternal catheters, under general anesthesia. Four rats were given 24 mu g FAM-labeled ASODNs while the others were given a saline control injection. After a 24 h observation period, rats were sacrificed by barbiturate overdose, and their brains were studied. Results. For all cell lines, fluorescence was seen to increase with increasing ASODNs concentration. Cells treated in similar fashion were also analyzed by flow cytometry to graphically illustrate the differing fluorescence. Multiple glioma cell lines were tested, with similar results. c-Met ASODNs was found to be successfully incorporated from the media into cultured human glioma cells, even at concentrations as low as 2 mu M. In addition, maintenance of the pH-dependent green fluorescence color, as seen by immunofluorescence microscopy and by using flow cytometry, indicated that the FAM was not contained within lysosomes. Immunofluorescence microscopy and RT-PCR analysis showed decreases in c-Met expression with oligodeoxynucleotides treatment. Uptake into tumor cells was also demonstrated in vivo, with no detectable toxicity at concentrations exceeding expected therapeutic levels. Conclusion. These data are encouraging for further study of c-Met antisense oligodeoxynucleotides as a therapeutic modality for glioma. (c) 2007 Elsevier Inc. All rights reserved.