ER-stress activation in human hepatocellular cancer cells: an alternative death pathway induced by the pan-deacetylase inhibitor panobinostat

Abstract

Background: We previously showed that panobinostat, a novel pan-deacetylase inhibitor, has a potent apoptotic activity in vitro and induces growth delay of HCC xenografts in nude mice. Panobinostat is able to induce cell death in HepG2 (p53wt) and in Hep3B (p53null) cell lines that is not dependent on canonical apoptotic pathways. Here we analyse the effect on ER-stress by panobinostat treatment. Methods: HepG2 and Hep3B cells were cultured under standard conditions and treated for 48h with 0.1µM panobinostat. Sub-G1 events were quantified by flow cytometry after propidium iodide staining and verified by immunofluorescence of cytokeratin 18 cleavage. ER-stress factors, p21 and acetylated histones were evaluated by qRT-PCR and western blotting. Caspase-12 activity was determined using a fluorometric assay. Results: Treatment of both cell lines induced cell death as was shown by an increase in sub-G1-events. An increase of p21 transcript and protein level was shown and a high histone acetylation status was observed. The unfolded protein response is clarified by IRE1-alpha and the chaperone BIP transcripts that showed an increase after treatment in vitro and in xenografts. Neither HCC cell line showed an expression of the splicing variant IRE1-beta or variations in the level of ATF-4. Panobinostat causes an increased expression of Chop/GADD45 and its protein and a decrease of Xbp-1 in HepG2 cells. We also demonstrated the up-regulation of eIF2-alpha and its phosphorylated form after treatment with panobinostat in HepG2 and Hep3B cells. The level of PERK shows also an increase and a phosphorylated status of JNK and p38MAPK is clearly detected. Finally, an increase of caspase-12 activity is detected. Conclusion: The novel pan-DACi Panobinostat induces cell death in HCC cell lines. ER-stress and the unfolded protein respone (UPR) represent alternative pathways that drive cells to die, independent of their p53 status.