Impact of new targeted therapies on the regulation of p53-family and cell death behavior of hepatoma cells in hepatocellular carcinoma

Background:

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide and recognized as one of the most chemo-resistant tumor types. Treatment options for HCC are divided into surgical therapies and nonsurgical therapies, which may be liver-directed or systemic. Until this year, sorafenib, a multikinase-inhibitor, was the only oral systemic therapy for patients with non-resectable HCC. However, sorafenib is indicated only for patients with well-preserved liver function (Child-Pugh A) and with advanced tumors (BCLC-C) or earlier stage tumors progressing upon or unsuitable for loco-regional therapies. With the new EASL guideline novel therapeutic substances such as lenvatinib were recommended and admitted in July 2018, expanding the therapeutic repertoire for HCC. Tumor suppressor p53 is not only the most mutated gene in human cancer but – together with its family members p63 and p73 – also an essential regulator of cell death. Since the molecular effect mechanisms of novel HCC therapeutics are only partially understood, the aim of this study was to elucidate the pathways by which these drugs induce cell death in HCC.

Methods:

HepG2 human HCC cell line cells were incubated with serum concentrations of HCC-relevant therapeutics (lenvatinib 1.17µM, ramucirumab 3,48µM, bleomycin 2µM, regorafenib 5µM, sorafenib 4.5µM) for 24 – 72h. Cabozantinib was administered in 2x serum concentration (2µM). DMSO treatment served as control. Drug-induced effects on mRNA level regarding the p53 family and pro-/antiapoptotic members of the Bcl-2 family were evaluated by qPCR. Levels of p53 family proteins were determined by Western blot. Cell viability was analyzed by MTS-Assay. Cell death was measured via flow cytometry using DAPI/Annexin V staining.

Results:

Viability of HepG2 cells was reduced by almost 50% after treatment with bleomycin, sorafenib, regorafenib and cabozantinib for 48h. However, flow cytometry analysis revealed that sorafenib and regorafenib treated cells did not display an apoptotic phenotype. Incubation with sorafenib, regorafenib and cabozantinib resulted in increased protein levels of p53, whereas p63 and p73 remained unaffected. Nevertheless, caspase cleavage as a characteristic feature of apoptosis was not detected and mRNA levels of proapoptotic Bcl-2 family members (Bax, Bak) were unchanged after treatment with sorafenib, regorafenib, cabozantinib.

Conclusion:

Novel HCC therapeutics are capable to initiate cell death in hepatoma cells. However, although p53 is induced by sorafenib, regorafenib and cabozantinib, cells die by an apoptosis-independent pathway. These findings not only expand our knowledge concerning the mode of action of drugs administered in HCC, but also indicate that – apart from apoptosis – p53 is involved in other types of cell death of hepatoma cells.