Original Article
Copyright ©2009 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. Dec 21, 2009; 15(47): 5924-5935
Published online Dec 21, 2009. doi: 10.3748/wjg.15.5924
TRAIL-induced apoptosis of hepatocellular carcinoma cells is augmented by targeted therapies
Bruno Christian Koehler, Toni Urbanik, Binje Vick, Regina Johanna Boger, Steffen Heeger, Peter R Galle, Marcus Schuchmann, Henning Schulze-Bergkamen
Bruno Christian Koehler, Toni Urbanik, Binje Vick, Regina Johanna Boger, Peter R Galle, Marcus Schuchmann, Henning Schulze-Bergkamen, First Department of Medicine, Johannes Gutenberg-University Mainz, 55101 Mainz, Germany
Steffen Heeger, Merck KGaA, MS-D Global Clinical Development Unit - Oncology, 64293 Darmstadt, Germany
Henning Schulze-Bergkamen, National Center of Tumor Diseases, Department of Medical Oncology, University Clinic of Heidelberg, 69120 Heidelberg, Germany
Author contributions: Koehler BC and Urbanik T performed the experiments and substantially contributed equally to the conception and design of the study, interpretation of the data and statistical analysis; Koehler BC, Urbanik T and Schulze-Bergkamen H wrote the manuscript; Vick B, Boger RJ, Schuchmann M, Schulze-Bergkamen H and Galle PR made substantial contribution to the design and conception of the study and interpretation of data; all authors read and approved the final manuscript; this study contains essential parts of the medical thesis work of Koehler BC.
Supported by Research grants from Merck KGaA, Darmstadt, Germany, to Schulze-Bergkamen H
Correspondence to: Henning Schulze-Bergkamen, MD, PhD, National Center of Tumor Diseases, Department of Medical Oncology, University Clinic of Heidelberg, Im Neuenheimer Feld 350, 69120 Heidelberg, Germany. henning.schulze@med.uni-heidelberg.de
Telephone: +49-6221-560 Fax: +49-6221-568815
Received: September 25, 2009
Revised: November 12, 2009
Accepted: November 19, 2009
Published online: December 21, 2009

AIM: To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors, in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand (TRAIL), on overcoming TRAIL resistance in hepatocellular carcinoma (HCC) and to study the efficacy of agonistic TRAIL antibodies, as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis.

METHODS: Surface expression of TRAIL receptors (TRAIL-R1-4) and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL were analyzed by flow cytometry and Western blotting, respectively. Knock-down of MCL-1 and BCL-xL was performed by transfecting specific small interfering RNAs. HCC cells were treated with kinase inhibitors and chemotherapeutic drugs. Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.

RESULTS: TRAIL-R1 and -R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2. However, treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates. Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002 [inhibition of phosphoinositol-3-kinase (PI3K)], AG1478 (epidermal growth factor receptor kinase), PD98059 (MEK1), rapamycin (mammalian target of rapamycin) and the multi-kinase inhibitor Sorafenib. Furthermore, the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL play a major role in TRAIL resistance: knock-down by RNA interference increased TRAIL-induced apoptosis of HCC cells. Additionally, knock-down of MCL-1 and BCL-xL led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K.

CONCLUSION: Our data identify the blockage of survival kinases, combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC.

Keywords: Hepatocellular carcinoma, Apoptosis, Tumor necrosis factor-related apoptosis inducing ligand, BCL-xL, MCL-1, 5-fluorouracil, Doxorubicin, Sorafenib, Phosphoinositol-3-kinase, (Mitogen-activated protein kinase)/(extracellular signal regulated kinase) kinase, c-Jun N-terminal kinase