Induction of apoptosis in hepatocellular carcinoma by a novel combination of histone deacetylase inhibitor LBH589

Background and Aims:

Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver and the third leading cause of cancer deaths worldwide. HCC cells show a high apoptotic capacity in earlier stages of carcinogenesis, whereas in advanced stages they gradually develop apoptosis resistance. This anti-apoptotic phenotype is associated with development and progression of HCC. Due to its chemoresistance, novel treatment options for HCC are in urgent demand. Histone deacetylase inhibitors promote apoptosis via the intrinsic apoptotic pathway, but at the same time sensitize tumor cells to death ligands that activate the extrinsic apoptotic pathway. The chemotherapeutic drug bleomycin causes single- and double-strande DNA breaks and produces reactive oxidative species leading to cell death. The aim of this study is to identify novel drug combinations in order to induce cell death in HCC cell lines and thus to extend the therapeutic repertoire for HCC.

Method:

The human hepatoma cell line HepG2 was incubated with serum concentrations of histone deacetylase inhibitor LBH589 (0.1 – 5µM) and bleomycin (2 – 10µM). DMSO treatment served as control. Cell death was measured after 12 and 24h via flow cytometry using DAPI/Annexin V-APC staining. The caspase Inhibitor Z-VAD-FMK and the RIPK1 inhibitor Nec-1 were added to investigate the effects of treatment combinations on cell death. Caspase cleavage as well as levels of pro-/anti-apoptotic members of the Bcl-2 family were determined by Western blot.

Results:

24h treatment with 0.1µM LBH589 induced a strong increase in cell death of up to 6.5-fold compared to the controls. 2µM bleomycin resulted in an increase of cell death of up to 4.5-fold. After incubation with a combination of LBH589 0.1µM and bleomycin 2µM cell death rates were increased to up to 14.8-fold, indicating a synergistic effect. Cell death could effectively be blocked by Z-VAD-FMK indicating induction of apoptosis. Incubation with LBH589, bleomycin and the combination of both therapeutics resulted in a cleavage of Caspases-3,-8,-9 and PARP, as well as in a downregulation of Bcl-XL as features of apoptosis.

Conclusion:

Our study demonstrates that novel combinations of already approved drugs like LBH589 and bleomycin could improve treatment options for HCC. Both drugs target different cellular pathways leading to synergistic effects and massive induction of apoptosis.