Effect of interferon-γ and tumor necrosis factor-α on hepatitis B virus following lamivudine treatment

AIM: To evaluate anti-hepatitis B virus (HBV) activity and cytotoxicity of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) following lamivudine treatment of HepG2.2.15 cells. METHODS: HepG2.2.15 cells were treated with 2 mu mol/L lamivudine for 16 d (lamivudine group), cultured for 10 d, followed by 5 ng/mL TNF-alpha and 1000 U/mL IFN-gamma for 6 d (cytokine group), or treated with 2 mu mol/L lamivudine for 10 d followed by 5 ng/mL TNF-alpha and 1000 U/mL IFN-gamma for 6 d (sequential group), or cultured without additions for 16 d (control group). Intracellular DNA was extracted from 3 x 10(5) HepG2.2.15 cells from each group. The extracted DNA was further purified with mung bean nuclease to remove HBV relaxed circular DNA that may have remained. Both HBV covalently closed circular DNA (cccDNA) and HBV DNA were examined with real-time polymerase chain reaction. The titers of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were quantified with enzyme-linked immunosorbent assay. Cell viability was measured with the cell counting kit-8 assay. RESULTS: Compared to lannivudine alone (22.63% +/- 0.12%), both sequential (51.50% +/- 0.17%, P = 0.034) and cytokine treatment (49.66% +/- 0.06%, P = 0.041) showed a stronger inhibition of HBV cccDNA; the difference between the sequential and cytokine groups was not statistically significant (51.50% +/- 0.17% vs 49.66% +/- 0.06%, P = 0.88). The sequential group showed less inhibition of HBV DNA replication than the lamivudine group (67.47% +/- 0.02% vs 82.48% +/- 0.05%, P = 0.014); the difference between the sequential and cytokine groups was not statistically significant (67.47% +/- 0.02% vs 57.45% +/- 0.07%, P = 0.071). The levels of HBsAg and HBeAg were significantly decreased in the sequential treatment group compared to the other groups [HBsAg: 3.48 +/- 0.04 (control), 3.09 +/- 0.08 (lamivudine), 2.55 +/- 0.13 (cytokine), 2.32 +/- 0.08 (sequential), P = 0.042 for each between-group comparison; HBeAg: 3.48 +/- 0.01 (control), 3.08 +/- 0.08 (lamivudine), 2.57 +/- 0.15 (cytokine), 2.34 +/- 0.12 (sequential), P = 0.048 for each between-group comparison]. Cell viability in the cytokine group was reduced to 58.03% +/- 8.03% compared with control cells (58.03% +/- 8.03% vs 100%, P = 0.000). Lamivudine pretreatment significantly reduced IFN-gamma + TNF-alpha-mediated toxicity of HepG2.2.15 cells [85.82% +/- 5.43% (sequential) vs 58.03% +/- 8.03% (cytokine), P = 0.002]. CONCLUSION: Sequential treatment overcame the lower ability of lannivudine alone to inhibit cccDNA and precluded the aggressive cytotoxicity involving IFN-gamma and TNF-alpha by decreasing the viral load. (c) 2012 Baishideng. All rights reserved.