HBx-induced reactive oxygen species activates hepatocellular carcinogenesis via dysregulation of PTEN/Akt pathway

Figure 1 Effect of hepatitis B virus X-protein on induction of aberrant cell growth. A: Cell growth analysis by crystal violet staining and A_{550 nm} detection. 10⁴ cells were seeded, and Stained at 1, 5, 6, 7 and 8 d after seeding. Values represent mean ± SD (n = 3). ^bP≤ 0.001 compared with mock transfectants; B: Morphology of the cells was observed by optical microscopy. HBx: Hepatitis B virus X-protein.

Figure 2 Effect of hepatitis B virus X-protein on activation of the Akt pathway. A: Activation of Akt pathway was examined by Western blotting with liver tissue extracts from 6-, 11- and 13-mo-old hepatitis B virus X-protein (HBx) transgenic and wild-type mice; B: Western blotting was also performed on extracts from stable HepG2-Mock and HBx cell lines.

Figure 3 Effect of hepatitis B virus X-protein-induced endogenous reactive oxygen species and phosphatase and tensin homolog oxidation. Endogenous reactive oxygen species (ROS) level was examined by flow cytometry. A, B: Increased production of ROS in hepatitis B virus X-protein (HBx) primary hepatocytes compared to the wild-type hepatocytes and HepG2-HBx compared to the Mock cells. Oxidized phosphatase and tensin homolog (PTEN) was detected by N-ethylmaleimide alkylation, and non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis; C: In the upper panel, 20 μg protein was loaded, and for the lower panel, 50 μg was loaded; D: Parallel experiments were performed with extracts from HepG2-Mock and HBx cell lines.

Figure 4 Effect of reactive oxygen species on phosphatase and tensin homolog oxidation, Akt pathway and hepatitis B virus X-protein expression. A, B: H₂O₂ treatment induced phosphatase and tensin homolog (PTEN) oxidation and activation of Akt pathway (increased relative p-Akt/total Akt ratio, cyclin D1 expression). Reactive oxygen species (ROS) scavenging through N-acetylcysteine (NAC) treatment reduced PTEN oxidation and Akt pathway; C: ROS effect on hepatitis B virus X-protein (HBx) expression, by quantitative reverse transcriptase polymerase chain reaction; D: Proposed scheme for ROS effect for activating Akt pathway via PTEN oxidation in HBx-induced hepatocarcinogenesis.