Ribavirin and alpha interferon enhance death receptor-mediated apoptosis and caspase activation in human hepatoma cells - PubMed (original) (raw)

Ribavirin and alpha interferon enhance death receptor-mediated apoptosis and caspase activation in human hepatoma cells

Stephan F Schlosser et al. Antimicrob Agents Chemother. 2003 Jun.

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 occurring 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.

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Figures

FIG. 1.

FIG. 1.

IFN and ribavirin induce apoptosis in HepG2 cells. HepG2 cells were incubated for 36 h with the indicated stimuli. Subsequently, nuclei were isolated by hypotonic lysis, stained with propidium iodide, and analyzed by flow cytometry for hypodiploid DNA content as an indicator of apoptosis. A total of 8,000 nuclei was counted per data point. The incidence of nuclei with hypodiploid DNA content is expressed as a percentage of the total number of nuclei. (A) IFN only; (B) ribavirin only; (C) 10 kU of IFN per ml plus ribavirin as indicated on the x axis. Values are means and standard deviations of triplicate determinations. For comparison, 0.25 μg of anti-CD95 per ml in combination with CHX (an inhibitor of translation) would result in 90 to 100% apoptosis after 24 h.

FIG. 2.

FIG. 2.

IFN and ribavirin enhance anti-CD95-induced apoptosis in HepG2 cells. HepG2 cells were pretreated for 24 h with the indicated stimuli and then stimulated for 12 h with 0.25 μg of anti-CD95 per ml. Subsequently, nuclei were isolated by hypotonic lysis, stained with propidium iodide, and analyzed by flow cytometry for hypodiploid DNA content as an indicator of apoptosis. A total of 8,000 nuclei was counted per data point. The incidence of nuclei with hypodiploid DNA content is expressed as a percentage of the total number of nuclei. (A) IFN only; (B) ribavirin only; (C) 10 kU of IFN per ml plus ribavirin as indicated on the x axis. Values are means and standard deviations of triplicate determinations. For comparison, the same dose of anti-CD95 in combination with CHX would result in 90 to 100% apoptosis after 24 h.

FIG. 3.

FIG. 3.

IFN and ribavirin induce DEVDase activity in HepG2 cells. Lysates of HepG2 cells were assayed with DEVD-AMC after incubation for 24 h with the indicated stimuli. The slope of the AMC release was measured fluorometrically and expressed as fold AMC release in unstimulated cells. (A) IFN only; (B) ribavirin only; (C) 10 kU of IFN per ml plus ribavirin as indicated on the x axis; (D) 100 kU of IFN per ml plus ribavirin as indicated on the x axis. Values are means and standard deviations of triplicate determinations. Representative plots from two independent experiments are shown.

FIG. 4.

FIG. 4.

IFN and ribavirin enhance anti-CD95-induced caspase activity in HepG2 cells. Lysates of HepG2 cells were assayed for caspase-3-like activity by using DEVD-AMC after preincubation for 24 h with the indicated stimuli and subsequent stimulation for 4 h with 0.25 μg of anti-CD95 per ml. The slope of the AMC release was measured fluorometrically and is expressed as fold AMC release in unstimulated cells. (A) IFN only; (B) ribavirin only; (C) 10 kU of IFN per ml plus ribavirin as indicated on the x axis; (D) 100 kU of IFN per ml plus ribavirin as indicated on the x axis. Values are means and standard deviations of triplicate determinations. Representative plots from two independent experiments are shown.

FIG. 5.

FIG. 5.

Pretreatment with IFN and ribavirin enhances anti-CD95-induced cleavage of PARP. HepG2 cells were pretreated for 24 h with medium (lanes 1 to 3), ribavirin (lanes 4 and 5), IFN (lanes 6 and 7), or the combination of ribavirin and IFN (lanes 8 and 9). Subsequently the cells were stimulated for 4 h with medium (lane 3), 0.25 μg of anti-CD95 per ml (lanes 1, 2, and 4 to 9), or anti-CD95 plus CHX (lane 1) as a positive control. Cell lysates were then assayed for PARP by immunoblot analysis. Uncleaved PARP is indicated by the open arrowhead, and cleavage products are indicated by the solid closed arrowhead. Note that the nonspecific band above 115 kDa serves as the loading control.

FIG. 6.

FIG. 6.

Pretreatment with IFN and ribavirin enhances anti-CD95-induced caspase-3 activation. Representative photomicrographs of HepG2 cells following stimulation with 0.25 μg of anti-CD95 per ml for 4 h and subsequent immunocytochemical detection of active caspase-3 are shown. (A) No pretreatment. (B) Pretreatment with 105 U of IFN per ml for 24 h. (C) Pretreatment with 103 μM ribavirin for 24 h. (D) Pretreatment with 105 U of IFN per ml plus 103 μM ribavirin for 24 h.

FIG. 7.

FIG. 7.

Pretreatment with IFN and ribavirin enhances anti-CD95-induced caspase-3 activation. This figure shows a semiquantitation of the photomicrographs in Fig. 6. Eight randomly selected microscopic fields (area of interest) per treatment were selected, and brown cells corresponding to cells stained with the specific antibody against active caspase-3 were counted.

FIG. 8.

FIG. 8.

Pretreatment with IFN and ribavirin enhances anti-CD95-induced cleavage of caspase-3, caspase-7, and caspase-8. HepG2 cells were pretreated for 24 h with medium (lanes 1 to 3), ribavirin (lanes 4 and 5), IFN (lanes 6 and 7), or the combination of ribavirin and IFN (lanes 8 and 9). Subsequently the cells were stimulated for 4 h with medium (lane 3), 0.25 μg of anti-CD95 (lanes 1, 2, and 4 to 9), or anti-CD95 plus CHX (lane 1) as a positive control. Cell lysates were then assayed by immunoblot analysis for caspase-3 (A), caspase-7 (B), and caspase-8 (C). Uncleaved procaspases are indicated by open arrowheads, and cleavage products are indicated by solid arrowheads. Note that procaspase-8 is not shown; instead, another nonspecific band from the same blot (NS) is shown; this band serves as a loading control.

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