Combining targeted therapies with HDAC inhibitors to tackle the p53 signaling pathway in hepatocellular carcinoma

Question The incidence of hepatocellular carcinoma (HCC) is increasing. Despite the progress in treatment options for HCC, drug therapies are often not sufficiently effective and associated with several side effects in patients with liver cirrhosis.

Preliminary work of our research group revealed a Mcl-1-dependent synergism of the multi-kinase inhibitor Sorafenib and the histone deacetylase inhibitor (HDACi) Vorinostat in the treatment of cutaneous T-cell lymphoma. Aim of this project was to evaluate whether such a synergistic effect can also be achieved in HCC by a combined treatment with Regorafenib and the HDACi Panobinostat. Panobinostat inhibits proliferation of malignant cells and is already approved for treatment of multiple myeloma.

Methods HepG2 and Huh7 hepatoma cells were incubated with Regorafenib (1-20 µM), with Panobinostat (5-20 nM) and with combinations of both therapeutics for 24-120 h. Cell viability was investigated by MTS assay and cell death was determined by flow cytometry after 7-AAD/FITC-Annexin V staining. Levels of p53 family were investigated by western blotting.

Results Panobinostat increased cell death induced by Regorafenib, both in HepG2 and in Huh7 cells. However, in Huh7 cells higher doses and longer incubation time were required for comparable effects. Monotherapy of HepG2 cells with Regorafenib (5µM) or Panobinostat (5 nM) led to a reduction of cell viability by 10 % after 72 h. In contrast, after combined treatment viability was reduced by 20 % after 72 h. Of note, treatment with Regorafenib alone and in combination with Panobinostat dose-dependently increased levels of p53 family proteins in HepG2. In Huh7, Regorafenib (5 µM) reduced cell viability by 20 % after 96 h. Additional treatment with Panobinostat (20 nM) led to a reduction by 40 %.

Enhancement of regorafenib-mediated cytotoxic effects by Panobinostat was shown by flow cytometry. Specific cell death of HepG2 cells after monotreatment with Regorafenib (5 µM) was about 24 % after 48 h, and about 42 % after 72 h. Combined treatment with Regorafenib (5 µM) and Panobinostat (5 nM) resulted in 41 % cell death after 48 h and 68 % after 72 h.

Conclusions Combination of the current targeted therapy Regorafenib with the HDACi Panobinostat increased the effectiveness of therapy in HepG2 and Huh7 cells. Based on these data, the long-term goal is to develop a form of therapy that allows a dose reduction of Regorafenib with the same outcome and reduced side effects.

Publikationsverlauf

Artikel online veröffentlicht:
04. Januar 2021