TRAIL-induced apoptosis of hepatocellular carcinoma cells is augmented by targeted therapies.
OBJECTIVE
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-x(L) were analyzed by flow cytometry and Western blotting, respectively. Knock-down of MCL-1 and BCL-x(L) 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-x(L) 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-x(L) led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K.


CONCLUSIONS
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.
Koehler
Bruno-Christian
BC
First Department of Medicine, Johannes Gutenberg-University Mainz, 55101 Mainz, Germany.
Urbanik
Toni
T
Vick
Binje
B
Boger
Regina-Johanna
RJ
Heeger
Steffen
S
Galle
Peter-R
PR
Schuchmann
Marcus
M
Schulze-Bergkamen
Henning
H
D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use; D017209:Apoptosis / Q000502:physiology*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000627:therapeutic use; D004791:Enzyme Inhibitors / Q000627:therapeutic use; D005472:Fluorouracil / Q000627:therapeutic use; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism*; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
D000970:Antineoplastic Agents; C494811:BCL2L1 protein, human; D004791:Enzyme Inhibitors; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053221:TNF-Related Apoptosis-Inducing Ligand; D051020:bcl-X Protein; D004317:Doxorubicin; D005472:Fluorouracil