Z Gastroenterol 2009; 47 - P3_11
DOI: 10.1055/s-0029-1191868

The novel histonedeacetylase inhibitor Panobinostat induces alternative pathways of apoptosis in hepatocellular carcinoma models

P Di Fazio 1, R Schneider-Stock 2, T Wissniowski 1, S Gahr 3, D Neureiter 4, M Ocker 3
  • 1Medizinische Klinik 1, Gastroenterologie und Hepatologie, Erlangen
  • 2Institut für Pathologie der Universität Magdeburg
  • 3Hepatologisches Forschungslabor, Medizinische Klinik I mit Poliklinik, Universität Erlangen-Nürnberg
  • 4Institut für Pathologie, Salzburger Landeskliniken, Österreich

Aims: Inhibitors of histone deacetylases (HDACi) represent a novel therapeutic option for human cancer diseases. The novel pan-HDACi Panobinostat (LBH589) induces apoptosis in human hepatocellular tumor cells in vitro and in xenograft models in vivo. Here, we analysed the underlying signalling pathways in more detail.

Methods: Human hepatocellular carcinoma (HCC) cell lines HepG2 (p53wt) and Hep3b (p53null) were cultured under standard conditions and treated with 0.1 or 0.01µM Panobinostat. Apoptosis was quantified by flow cytometry and verified by immunofluorescence of cytokeratin 18 cleavage. Mitochondrial transmembrane potential was detected by DiOC6 staining. Apoptotic pathways were analysed by quantitative RT-PCR and by Western blotting. Activity of caspases was determined using Caspase Glo. miRNA expression was determined using the miScript system and quantitative RT-PCR:

Results: Incubation of both HCC cell lines induced apoptosis as was shown by an increase in sub-G1-events in flow cytometry. Apoptotic cell death was verified by activation of the executioner caspase 3. No changes in ΔΨm or in proteins of the bax/bcl-2 family were detected. Only in p53null cells a strong activation of caspase 8 was observed, which was paralleled by an increase of the TRAIL receptor hDR5. Blocking hDR5 did not influence apoptosis levels while a pan-caspase inhibitor significantly lowered apoptosis and blocked activation of caspase 8. In p53wt HepG2 cells, the unfolded protein response (UPR) was activated as was shown by induction of xbp1 and Chop with subsequent activation of caspase 12. The tumor suppressor miRNA let-7b was strongly induced in HepG2 but not in Hep3B with a subsequent downregulation of target genes like HMGA2.

Conclusion: The novel HDACi Panobinostat induces apoptotic cell death in HCC cell lines. Dependent on their p53 status, novel alternative pathways of apoptosis induction (e.g. UPR, miRNA) or the extrinsic pathway can be activated.

HDAC - UPR - apoptosis - miRNA - p53