title: Effect of interferon-γ and tumor necrosis factor-α on hepatitis B virus following lamivudine treatment.
abstract: OBJECTIVE
  To evaluate anti-hepatitis B virus (HBV) activity and cytotoxicity of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) following lamivudine treatment of HepG2.2.15 cells.
  
  
  METHODS
  HepG2.2.15 cells were treated with 2 μmol/L lamivudine for 16 d (lamivudine group), cultured for 10 d, followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ for 6 d (cytokine group), or treated with 2 μmol/L lamivudine for 10 d followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ for 6 d (sequential group), or cultured without additions for 16 d (control group). Intracellular DNA was extracted from 3 × 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 lamivudine 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-γ + TNF-α-mediated toxicity of HepG2.2.15 cells [85.82% ± 5.43% (sequential) vs 58.03% ± 8.03% (cytokine), P = 0.002].
  
  
  CONCLUSIONS
  Sequential treatment overcame the lower ability of lamivudine alone to inhibit cccDNA and precluded the aggressive cytotoxicity involving IFN-γ and TNF-α by decreasing the viral load.
authors: Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, Shanghai 200032, China. shihongcn2000@yahoo.com.cn
authors: Hong
authors: H
authors: Shi
authors: Lu
authors: L
authors: Lu
authors: Ning-Ping
authors: NP
authors: Zhang
authors: Shun-Cai
authors: SC
authors: Zhang
authors: Xi-Zhong
authors: XZ
authors: Shen
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D004279:DNA, Viral / Q000096:biosynthesis / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D056945:Hep G2 Cells; D006514:Hepatitis B Surface Antigens / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000378:metabolism; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology / Q000821:virology; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D019259:Lamivudine / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D019562:Viral Load; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D019259:Lamivudine; D007371:Interferon-gamma