S-adenosyl methionine improves early viral responses and interferon-stimulated gene induction in hepatitis C nonresponders

Abstract

Background & aims: Less than half of patients infected with hepatitis C virus (HCV) achieve sustained viral clearance after pegylated interferon (peginterferon) and ribavirin therapy. S-adenosyl methionine (SAMe) improves interferon signaling in cell culture. We assessed the effect of SAMe on the kinetics of the early antiviral response and interferon signaling in nonresponders to previous antiviral therapy and investigated the mechanisms involved.

Methods: Nonresponders with HCV genotype 1 were given peginterferon alfa-2a and ribavirin for 2 weeks (course A, baseline/control). After 1 month, patients received SAMe (1600 mg daily) for 2 weeks and then peginterferon and ribavirin for 48 weeks (course B; completed by 21 of 24 patients). Viral kinetics and interferon-stimulated gene (ISG) expression in peripheral blood mononuclear cells (PBMCs) were compared between courses.

Results: The decrease in HCV RNA from 0 to 48 hours (phase 1) was similar with and without SAMe. However, the second phase slope of viral decline was improved with SAMe (course A, 0.11 ± 0.04 log(10) IU/mL/wk; course B, 0.27 ± 0.06; P = .009); 11 patients (53%) achieved an early virological response, and 10 (48%) had undetectable HCV RNA by week 24. Induction of ISGs in PBMCs was significantly greater during course B. In cultured cells, SAMe increased induction of ISGs and the antiviral effects of interferon by increasing STAT1 methylation, possibly affecting STAT1-DNA binding.

Conclusions: The addition of SAMe to peginterferon and ribavirin improves the early viral kinetics and increases ISG induction in nonresponders to previous therapy. SAMe might be a useful adjunct to peginterferon-based therapies in chronic HCV infection.

Trial registration: ClinicalTrials.gov NCT00475176.

Figure 1. Effects of SAMe on HCV RNA levels and on first and second phase kinetic responses to peginterferon and ribavirin

Viral kinetics were compared between Course A (peginterferon and ribavirin alone) and Course B (peginterferon, ribavirin and SAMe) showing the effect of SAMe on a) the first phase viral decline, b) the average second phase slope and c) on HCV RNA levels as monotherapy. P-values are based on Wilcoxon matched pairs test.

Figure 2. Effects of SAMe on ISG induction in PBMCs of patients receiving peginterferon and ribavirin

Expression of a) ISG15, b) Mx1 and c) RSAD2 mRNA levels in PBMC were compared between Course A (peginterferon and ribavirin alone) and Course B (peginterferon, ribavirin and SAMe) at six time points. Values reflect the average fold-induction compared to the baseline expression [Time 0] for each patient. The area under the curve (AUC) reflects the mean fold-induction over all time-points measured. Mean AUC values were compared using the non-parametric Wilcoxon matched pairs test. d) Protein expression of RSAD2, DMA and actin in PBMC was compared between baseline and 24 hours in Course A and after SAMe loading at baseline and 24 hrs in Course B. STAT1 methylation was assessed by IP for STAT1 followed by WB for DMA. Results are shown for a representative patient. Relative units (RU) compared using time 0 for each Course as baseline. * p<0.05 for comparison.

Figure 3. Effects of SAMe on ISG induction in-vitro

a) Huh7.5.1 cells were treated with SAMe alone or SAMe combined with interferon-α (50 IU/mL). Cells were incubated with SAMe for 2 hours before addition of interferon and were harvested for assessment of gene induction 4 hours after interferon treatment. ISG expression (Mx1) was evaluated by real-time PCR. b) Huh7.5.1 cells infected with the JFH1 clone of HCV (for 24 hours) were subjected to the same treatment using SAMe alone or combined with interferon. Note the scales of Mx1 induction are different with SAMe alone and SAMe + interferon. Mean results from 3 independent experiments shown. *p<0.05 for comparison with interferon-α alone.

Figure 4. Effects of addition of SAMe to interferon on HCV RNA levels in-vitro

Huh7.5.1 cells were infected with the JFH1 HCV for 24 hours and then treated with SAMe alone or SAMe (for 2 hours) and interferon-α (50 IU/mL). Cells were harvested at different time-points after interferon treatment and intracellular HCV RNA was measured by real-time PCR. Mean results from 3 independent experiments shown. *p<0.05 for comparison with interferon-α alone.

Figure 5. Effects of SAMe on STAT1 methylation

a) Huh7.5.1 cells were treated with SAMe (800nM) for 2 hours, interferon-α for 1 hour or the combination (SAMe pretreatment followed by interferon-α) and then harvested in RIPA buffer. IP was performed with anti-STAT1, anti-PIAS1 or anti-DMA antibodies and then WB was performed with each individual antibody. The effect of SAMe treatment with or without interferon on STAT1 methylation (first panel), PIAS1 methylation (second panel) and the interaction between PIAS1 and STAT1 (third panel) is shown. b) The experiment was repeated in the presence of HCV infection. IP, immunoprecipitation; WB, western blot; RU, relative units.