In vitro gene targeting in human Hepatoblastoma

Abstract

Poor treatment results in advanced hepatoblastoma (HB) made alternative treatment approaches desirable. Gene-directed tumor therapy is increasingly investigated in different malignancies. The aim of this study was to analyze possible alternatives of gene transfer into HB cells and to study therapeutic applications based on different strategies. Liposomal transfection of HB cells was assessed using liver-specific promoters, and adenovirus and Sendai virus transductions were performed in vitro. Transfer efficiencies were measured via flow cytometry determining expression of vector-encoded marker gene green fluorescent protein. Gene silencing of the anti-apoptotic bcl-2 gene in HUH6 cells was performed using lipofection of small interfering RNA (siRNA). Additionally, suicide gene therapy was carried out through a yeast-derived cytosine deaminase (YCD)-combined yeast uracil phosphoribosyltransferase (YUPRT)-based adenovirus-mediated gene transfer, leading to a potent intracellular prodrug transformation of 5-fluorocytosine into 5-fluorouracil. Treatment efficiencies were monitored via MTT viability assay. Highest gene transfer rates (86%) were observed using adenovirus transduction. We furthermore observed a significant therapeutic effect of adenovirus-mediated YCD::YUPRT suicide gene transfer. Liposomal-mediated anti-bcl-2 siRNA transfer led to a significant improvement of cisplatin treatment in HUH6 cells. Liver-specific promoters were found to be strongly active in HUH6 cells (mixed HB-derived), but less active in HepT1 cells (embryonal HB-derived). Liposomal transfection and viral transduction are effective approaches to genetically manipulate HB cells in vitro. For the first time, we demonstrate a positive effect of siRNA gene silencing in this malignancy. Additionally, we successfully investigated a model of adenovirus-based suicide gene therapy in HB cell cultures. Our data strongly encourage further studies assessing these alternative treatment approaches.

Bcl-2 Gene Silencing in Pediatric Epithelial Liver Tumors

“…Whereas Feng postulated Bcl-2 as potential therapy target of the HCC cell line HepG2, Ikeguchi et al did not find a clinical importance of the Bcl-2/Bax ratio analyzing 53 patients with HCC [13,34]. Our own preliminary data indicated the possibility of improved treatment outcomes in HB through Bcl-2 knockdown [5].…”

“…The technique of RNA interference using siRNA has been established as an effective treatment approach against various malignancies including liver tumors in adults [5,6,24,39]. Although there exist alternative methods of cell transfection [40,41], we found the siRNA approach being effective and reliable for the intended purpose.…”

Optimizing vector application for gene transfer into human hepatoblastoma cells

“…As a general rule, the viral vectors are known to display higher transfer rates of therapeutic genes than lipofectants, as we recently confirmed for the HB gene transfer in a pilot study using adenoviral and Sendai viral vectors [26].…”

Emerging gene-directed anti-tumor strategies against human hepatoblastoma

“…HB were found to express differing transcriptional activities of synthetic liver-specific transcriptional control unit constructs according to the histological background [42]. These findings indicate the necessity for a purposeful selection of HB active promoter cassettes prior to their employment in future therapeutic approaches of HB treatment.…”

“…Initial studies on HB demonstrated the tumors' susceptibility to gene transfer, clone development and therapeutic approaches using liposomal transfection as well as viral vectors [41,42]. Systematic analyses of gene transfer systems revealed liposomal transfection using either the multi-component lipid based FuGENE 6™ transfection reagent (Roche Diagnostics), or the cationic lipid reagent Lipofectamine™ 2000 (Invitrogen) as advantageous in terms of handling and safety.…”