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, 16 (39), 4932-7

HBx-induced Reactive Oxygen Species Activates Hepatocellular Carcinogenesis via Dysregulation of PTEN/Akt Pathway

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HBx-induced Reactive Oxygen Species Activates Hepatocellular Carcinogenesis via Dysregulation of PTEN/Akt Pathway

Hye-Lin Ha et al. World J Gastroenterol.

Abstract

Aim: To investigate the role of hepatitis B virus X-protein (HBx)-induced reactive oxygen species (ROS) on liver carcinogenesis in HBx transgenic mice and HepG2-HBx cells.

Methods: Cell growth rate was analyzed, and through western blotting, mitogenic signaling was observed. Endogenous ROS from wild and HBx transgenic mice and HepG2-Mock and HBx cells were assayed by FACScalibur. Identification of oxidized and reduced phosphatase and tensin homolog (PTEN) was analyzed through N-ethylmaleimide alkylation, nonreducing electrophoresis.

Results: We observed that the cell-proliferation-related phosphoinositide 3-kinase/Akt pathway is activated by HBx in vivo and in vitro. Increased ROS were detected by HBx. Tumor suppressor PTEN, via dephosphorylation of Akt, was oxidized and inactivated by increased ROS. Increased oxidized PTEN activated the mitogenic pathway through over-activated Akt. However, treatment with ROS scavenger N-acetyl cysteine can reverse PTEN to a reduced form. Endogenously produced ROS also stimulated HBx expression.

Conclusion: HBx induced ROS promoted Akt pathways via oxidized inactive PTEN. HBx and ROS maintained a positive regulatory loop, which aggravated carcinogenesis.

Figures

Figure 1
Figure 1
Effect of hepatitis B virus X-protein on induction of aberrant cell growth. A: Cell growth analysis by crystal violet staining and A550 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
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
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
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.

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