Sensitization of hepatocytes by methyltransferase-inhibitor 5-azacytidine against cytokine-mediated cell death

Background 5-Azacytidine (5-aza-CR) was developed as an antineoplastic agent long before its inhibitory activity on DNA methyltransferases and thus its ability to reverse aberrant gene silencing became evident. Today, 5-aza-CR is the first DNA hypomethylating agent approved for treatment of myelodysplastic syndromes and represents a promising drug for epigenetic cancer therapy. However, 5-aza-CR is contraindicated for patients with pre-existing liver diseases due to adverse liver effects for yet unknown reasons.

Aims: For a comprehensive understanding of the pharmacodynamic properties of 5-aza-CR it is mandatory to elucidate and profile this sensitizing effect

Results: In a time- and concentration-dependent manner, hepatocytes, i.e. primary murine and human hepatocytes and HepG2 cells, became highly sensitive towards death receptor-mediated cell death (induced either by CD95L, TRAIL or TNF) when pre-treated with sub-toxic concentrations of 5-aza-CR. 5-aza-CR-enabled hepatocyte cell death was characterized by classical hallmarks of apoptosis such as membrane blebbing, chromatin condensation and exposure of phosphatidyl serine on the outer membrane. Futhermore we demonstrated an upregulation of pro-apoptotic regulator proteins p53 and BAX in a time- and concentration-dependent manner in 5-aza-CR treated HepG2 cells. UV radiation is a DNA-damaging stimulus that activates a p53/Bax-dependent apoptotic response in this system. We also detected an increased cytotoxicity as well as caspase-activity after irradiation with UV radiation in the presence of 5-azac-CR..

Moreover, in the presence of 5-aza-CR, activation of downstream caspases was markedly enhanced when the extrinsic pathway of apoptosis was activated by heterologous and transient expression of caspase–8. In contrast, artificial activation of the intrinsic pathway of apoptosis within S–100 cytosolic fractions of HepG2 via dATP/Cytochrom C failed to induce upregulation of caspase activity.

Conclusions: This study reveals that 5-aza-CR sensitizes hepatocytes against death receptor agonists. As a mechanistic rationale, we provide evidence for a p53/Bax dependent sensitizing mechanism acting in-between the death inducing signaling complex (DISC) and above the mitochondrial level. In summary, our findings offer a mechanistic explanation for the adverse hepatotoxic properties of 5-aza-CR observed in patients with pre-existing liver disorders.

HepG2 - TNF - apoptosis - death receptors