title: TRAIL-induced apoptosis of hepatocellular carcinoma cells is augmented by targeted therapies.
abstract: 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.
authors.affiliation: First Department of Medicine, Johannes Gutenberg-University Mainz, 55101 Mainz, Germany.
authors.forename: Bruno-Christian
authors.initials: BC
authors.lastname: Koehler
authors.forename: Toni
authors.initials: T
authors.lastname: Urbanik
authors.forename: Binje
authors.initials: B
authors.lastname: Vick
authors.forename: Regina-Johanna
authors.initials: RJ
authors.lastname: Boger
authors.forename: Steffen
authors.initials: S
authors.lastname: Heeger
authors.forename: Peter-R
authors.initials: PR
authors.lastname: Galle
authors.forename: Marcus
authors.initials: M
authors.lastname: Schuchmann
authors.forename: Henning
authors.initials: H
authors.lastname: Schulze-Bergkamen
mesh_terms: 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
chemical_list: 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