Inhibitory effects and the molecular mechanism of tenofovir disoproxil fumarate and adefovir dipivoxil on proliferation of hepatic stellate cells

Abstract

Background: It has been reported that long-term suppression of replication of hepatitis B virus (HBV) with nucleotide analogues such as tenofovir (TDF) or adefovir (ADV) is associated with regression of advanced hepatic fibrosis and reversal of cirrhosis. These effects are considered mainly due to decreased liver injury caused by HBV, but direct anti-fibrotic effects of these drugs might be associated. We planned to investigate the effect of these drugs on the hepatic stellate cells which are key driver cells causing hepatic fibrosis.

Methods: The immortalized human hepatic stellate cell line, LX-2 was cultured. The cells were treated with control, TDF and ADV for 24 and 48 hours. Cellular DNA synthesis was evaluated by measuring BrdU incorporation. Distribution of cell cycle was determined by propidium iodide (PI) staining and flow cytometry. Expression of proteins including cell cycle regulators (p21, p27, cyclin D1),NF-kB, TGF-β, α-smooth muscle actin (α-SMA), Erk1/2 andpErk1/2 were measured in Western blot analysis.

Results: InBrdU-proliferation assay, both of two drugs effectively inhibited the proliferation dependent on the concentration. All of two drugs markedly reduced p27, p21 and induced cyclin D1 compared to control. In addition, the cell cycle arrest at the G2/Mphase was observed according to the concentration of two drugs by using flow cytometry. The amounts of NF-kB and TGF-βwere decreased by both of two drugs. No significant changes of two drugs on protein expression of α-SMA, Erk1/2 andpErk1/2 were observed.

Conclusions: TDF and ADF are capable of reducing proliferation of activated HSCs. We believe that the mechanism of anti-proliferative effect seems to be mediated by G2/M cell cycle arrest. Furthermore, both of two drugs suppressed NF-kB, TGF-β transduction signaling. These data suggest that TDF and ADF effectively block the proliferation and growth of activated HSCs, therefore both of two drugs would be associated with regression the liver fibrosis.