These data indicate that inhibition of TLR4 signaling increases susceptibility to DEN-induced carcinogenesis and progression of HCC through enhancing proliferation and attenuating liver cell death. The extent of DNA damage was analyzed by immunoperoxidase staining for 8-OHdG and the expression of γ-H2AX and H2AX in WT and TLR4 mutant liver tissue. We found that DEN insult resulted in a time-dependent increase in the expression of 8-OHdG and γ-H2AX in TLR4-deficient

liver tissue (Fig. 2A-D) but a time-dependent recovery in the WT liver tissue, which was confirmed by the analysis of γ-H2AX expression in liver tissue by confocal microscopy analysis (Fig. 2E,F). Similarly, TLR4mut mice showed a time-dependent gradual increase in the accumulation of ROS in liver tissue, but WT littermates showed a time-dependent gradual decrease in ROS production (Fig. 3A,B). Notably, the expression of DNA repair protein C646 Ku70 in the liver was significantly decreased in the DEN-treated

TLR4mut mice up to 60 days after DEN injection (Fig. 3C,D and Supporting Fig. 1G). The expression of Ku80, a partner of Ku70, was also decreased in the early days after DEN injection but gradually return to http://www.selleckchem.com/products/abt-199.html the basal level after 30 days after DEN injection (Fig. 3C,D). Consistent with these observations, the expression or activity of DNA-dependent repair kinase complex ATM-DNA-PKcs was attenuated in TLR4mut liver tissue compared with DEN-treated WT littermates (Fig. 3E,F and Supporting Fig. 1E,F). These data indicate that TLR4mut mice show a persistent increase in DNA damage response and ROS accumulation which associated with a suppressive expression of DNA repair proteins Ku70/80 and activity MCE of DNA repair protein kinase complex in DEN-treated TLR4mut liver tissue. To determine the potential role of immune cells in HCC development

in TLR4mut liver, the liver-infiltrating F4/80+ macrophages were examined in sham- or DEN-treated WT and TLR4mut livers. TLR4 mutation caused a marked decrease in the liver-filtrating F4/80+ macrophages compared with WT mice (Supporting Fig. 2A,B). TLR4 mediates a diversity of cellular functions by activating the MyD88-dependent apoptosis signal-regulating kinase 1 (ASK1)/p38 mitogen-activated protein kinase (p38 MAPK)/nuclear factor kappa BNF-κB signaling or MyD88-independent IRF3 pathway.24 The ASK1/p38 MAPK/NF-κB pathway is not only a major sensor of oxidative stress leading to programmed cell death, but it is also sufficient and necessary to induce cellular senescence against carcinogenesis after DNA damage and genomic instability.18 Moreover, cytokine production mediated by NF-κB activation plays important roles in triggering cell death and critically contributes to the cellular senescence against tumorigenesis.25-27 We found that TLR4 deficiency resulted in a broad-spectrum decline of immune responses to DEN-induced liver injury.