Combination of panobinostat and bleomycin induces apoptosis in Hepatocellular Carcinoma (HCC) through downregulation of Bcl-XL and Mcl-1

Background Hepatocellular carcinoma (HCC) is still a challenge in clinical practice, since HCC cells in advanced stages develop an apoptotic resistant phenotype being associated with development and progression of HCC. We evaluated the effect of combining histone deacetylase inhibitors and the chemotherapeutic agent bleomycin on cell death induction in HCC cells. Method: HepG2 cells were incubated with serum concentrations of histone deacetylase inhibitor panobinostat (0.1-5µM) and bleomycin (2-10µM). Cell death was detected after 12 and 24h by flow cytometry using DAPI/Annexin V-APC staining. The caspase inhibitor Z-VAD-FMK was added to determine the mode of cell death. Caspase cleavage as well as levels of pro-and anti-apoptotic members of the Bcl-2 family were analyzed by Western blot.

Results 24h treatment with 1μM panobinostat induced an increase in cell death of up to 25% compared to controls. 10µM bleomycin resulted in cell death induction of 35% of the HCC cells. After incubation with a combination of panobinostat and bleomycin cell death was increased by up to 60%, indicating a synergistic effect. Cell death could effectively be blocked by zVAD-FMK, suggesting apoptosis as the underlying mechanism. Panobinostat and bleomycin alone as well as the combination of both resulted in a cleavage of caspases-3,-8, and -9, a downregulation of the anti-apoptotic proteins Bcl-XL and Mcl-1 as well as a cleavage of PARP, confirming induction of apoptosis.

Conclusion Panobinostat and bleomycin target different cellular pathways leading to synergistic effects and pronounced induction of apoptosis in HCC, playing an important role for future therapies.

Publikationsverlauf

Artikel online veröffentlicht:
18. Januar 2023