Ribavirin and Alpha Interferon Enhance Death Receptor-Mediated Apoptosis and Caspase Activation in Human Hepatoma Cells

Abstract

The molecular mechanisms underlying the clinical effects of alpha interferon (IFN) and ribavirin are not understood. Elimination of infected cells occurs in part by cytotoxic T lymphocytes (CTLs) expressing CD95 ligand and thereby attacking target cells which are positive for the death receptor CD95. Since many viruses have evolved mechanisms to inhibit apoptosis, the opposite, namely, promotion of apoptosis, could be a strategy to strengthen the host antiviral response. In the present study, we have asked whether the antiviral substances IFN and ribavirin could support CD95-mediated apoptosis by interfering with the activation of caspases, a family of proteases known for their essential role in apoptosis. HepG2 cells, stimulated with the agonistic anti-CD95 antibody, served as a minimal model to mimic the CD95 stimulation ocurring during a CTL attack of target cells in vivo. Apoptosis was quantitated by flow cytometric detection of hypodiploid nuclei. Caspase activity was measured by cytofluorometry, immunocytochemistry, and immunoblot analysis. IFN and ribavirin sensitized HepG2 cells for CD95-mediated apoptosis. This effect was correlated with an increase in CD95-mediated caspase activation and enhanced cleavage of the caspase substrate poly(ADP-ribose) polymerase. Furthermore, the positive effect on CD95-mediated caspase activation by IFN and ribavirin was confirmed by immunocytochemistry for activated caspase-3 and by immunoblot detection of activated caspase-3, caspase-7, and caspase-8. Our data demonstrate that the antiviral substances IFN and ribavirin are able to sensitize for CD95-mediated apoptosis. IFN and ribavirin also enhance CD95-mediated caspase activation, which might in part be responsible for the apoptosis-promoting effect of these antiviral compounds.

Hepatic gene expression during treatment with peginterferon and ribavirin: Identifying molecular pathways for treatment response

“…Although there were mixed effects, caspase 8, the main activator of the Fas-mediated apoptosis pathway, was up-regulated in ribavirin-treated patients. This is in keeping with a previous report from Schlosser et al, 30 who showed that IFN and ribavirin treatment of Hep G2 cells resulted in increased caspase 8 expression and increased Fas-mediated apoptosis. The finding that apoptotic pathways are also up-regulated in responders to therapy suggests that apoptosis may be a critical component of the response to HCV treatment.…”

Metamorphosis inXenopus laevisis not associated with large-scale nuclear DNA content variation

“…Other investigators have observed that during apoptosis, chromatin condensation occurs, which results in reduced PI fluorescence (O'Brien et al, 1998). It has now become an acceptable practice to take advantage of the reduced PI fluorescence due to chromatin compaction to quantify apoptosis by observing the presence of hypodiploid nuclei using flow cytometry (Hess et al, 2001;Schlosser et al, 2003;Souza-Fagundes et al, 2003).…”

Apoptotic and anti-angiogenic strategies in liver and gastrointestinal malignancies

“…IFN binds to its cell surface receptor leading to the expression of a set of proteins that mediate the biological actions of IFN. The process is regulated by Janus kinases (JAK) and signal-transducing and activating factors of transcription (STAT) [47], thereby either affecting directly infected or uninfected cells or interfering indirectly with the regulation of the immune system [46]. In cells infected with RNA viruses, IFN induces the double-stranded RNA-dependent serine/ threonine protein kinase PKR, a key molecule with respect to the antiviral potency of IFN [46].…”

“…The process is regulated by Janus kinases (JAK) and signal-transducing and activating factors of transcription (STAT) [47], thereby either affecting directly infected or uninfected cells or interfering indirectly with the regulation of the immune system [46]. In cells infected with RNA viruses, IFN induces the double-stranded RNA-dependent serine/ threonine protein kinase PKR, a key molecule with respect to the antiviral potency of IFN [46]. Apart from this direct antiviral potency, cells overexpressing PKR are more sensitive to apoptosis, and it has been demonstrated that PKR can induce PARP cleavage and an upregulation of CD95 mRNA in NIH 3T3 cells [46].…”

“…The guanosine analog ribavirin (1-b-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is transported into almost all cell types of the body by sodium-dependent nucleoside transporters [45] and is capable of modulating cytokine profiles and the differentiation pattern of lymphocytes [46]. On the other hand, interferon (IFN) belongs to the group of acid-stable IFNs that are produced mainly by leukocytes and lymphoblasts.…”