[Frontiers in Bioscience E4, 2194-2202, January 1, 2012]

[Frontiers in Bioscience E4, 2194-2202, January 1, 2012]

Proteasome inhibition overcomes TRAIL resistance in human hepatoblastoma cells

Sorin Armeanu-Ebinger¹, Joerg Fuchs¹, Julia Wenz¹, Guido Seitz¹, Peter Ruck², Steven W Warmann¹

1Department of Pediatric Surgery, University Children's Hospital Tubingen, Hoppe-Seyler-Str. 3, 72076 Tubingen, Germany, ²Institute of Pathology, Hospital Leonberg, 71229 Leonberg, Germany

TABLE OF CONTENTS

1. Abstract
2. Background
3. Methods: 3.1. Tissue samples; 3.2. Cell lines and culture conditions; 3.3. Viability assay; 3.4. Transfection with siRNA against FLIP; 3.5. Immunofluorescence; 3.6. Detection of apoptosis; 3.7. Cytotoxicity assay; 3.8. Statistical analyses
4. Results: 4.1. Expression of TRAIL receptors on hepatoblastoma cells; 4.2. Effects of TRAIL incubation on hepatoblastoma cells; 4.3. Effects of FLIP inhibition on hepatoblastoma cells; 4.4. Inhibition of Bcl-2 does not induce sensitivity of hepatoblastoma cells to TRAIL; 4.5. Proteasome inhibition overcomes TRAIL resistance in hepatoblastoma cells
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is responsible for cell death in many cancer cells while being non-toxic for most normal cells. In this study, we investigated the role of TRAIL in human hepatoblastoma (HB) cells and analyzed different approaches to reverse TRAIL resistance in these tumors. Death receptors DR4 and DR5 expression was found on all analyzed primary HB samples and on the cell lines HuH6 and HepT1 by immunofluorescence staining. Recombinant TRAIL alone did not induce in vitro cytotoxicity. Decoy receptor blocking by antibodies led to moderate effects in HepT1 but not in HUH6 cells, whereas FLIP knock-down using siRNA rendered HUH6 cells but not HepT1 cells sensible to TRAIL. Bcl-2 inhibition with ABT-737 enhanced TRAIL-mediated apoptosis in all HB cells. Strongest cytotoxic TRAIL effects were seen in HB cell lines with synchronous proteasome inhibition using bortezomib. FLIP and Bcl-2 contributed to the TRAIL resistance in HB. Overcoming TRAIL resistance in HB by proteasome inhibitors has been identified a possible additive to improve treatment results in HB patients with drug resistant tumors.